Polypeptides and immunizing compositions containing gram positive polypeptides and methods of use

ABSTRACT

The present invention provides isolated polypeptides isolatable from a Staphylococcus spp. Also provided by the present invention are compositions that include one or more of the polypeptides, and methods for making and methods for using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/210,772, filed Mar. 23, 2009.

BACKGROUND

Gram-positive bacteria are a remarkably diverse group of organisms thatcause a variety of diseases in both humans and animals. Some of thepathogens recognized as important in human and/or animal health includebacteria belonging to the families of Corynebacteriaceae,Enterococcacae, Micrococcaceae, Mycobacteriaceae, Nocardiaceae, andPeptococcaceae, which include such bacterial species as Actinomycesspp., Bifidobacterium spp., Corynebacterium spp., Enterococcus spp.,Erysipelothrix spp., Eubacterium spp., Kytococcus spp., Lactobacillusspp., Micrococcus spp., Mobiluncus spp., Mycobacteria spp.,Peptostreptococcus spp., Propionibacterium spp., and Staphylococcus spp.These pathogens cause a multitude of clinical manifestations in manydifferent animal species. The treatment for such infections hashistorically been antibiotics that attack the common structures andfunctions of gram-positive organisms. However, many of the moreubiquitous gram-positive organisms have developed resistance to severalclasses of antibiotics, making treatment of infections difficult. Thewidespread use of antibiotics in the treatment of bacterial diseases inboth humans and food production animals is likely a major contributingfactor in the proliferation of antibiotic-resistant strains of manyspecies of gram-positive organisms. Therefore, there is a great need tofind different treatments that prevent or eliminate infections bygram-positive organisms in animals as well as humans.

Staphylococcal Infections in Agricultural Animals

In the agricultural industry a number of important diseases are causedby gram-positive organisms. Examples of clinical conditions caused bygram positive bacterial infections include, mastitis, septicemia,pneumonia, osteomyelitis, meningoencephalitis, lymphangitis, dermatitis,genital tract infections, metritis, perinatal disease, pituitaryabscesses, arthritis, bursitis, orchitis, cystitis and pyelonephritis,caseous lymphadenitis, tuberculosis, ulcerative lymphangitis,erysipelas, laminitis, tyzzer's disease, tetanus, botulism, enteritis,malignant edema, braxy, bacillary hemoglobinuria, enterotoxemia.Staphylococcus spp., in particular, are capable of infecting manydifferent species of agricultural animals and can cause enormouseconomic losses. For example, the United States dairy industry isestimated to lose approximately $185 per cow annually due to mastitis, adisease often caused by Staphylococcus aureus. Since there are 9.5million head of milking cows in the U.S., the annual cost of mastitis isapproximately $1.8 billion. This is approximately 10% of the total valueof farm milk sales, and about two-thirds of this loss is due to reducedmilk production in sub-clinically infected cows. Other losses are due todiscarded abnormal milk and milk withheld from cows treated withantibiotic, costs of early replacement of affected cows, reduced salevalue of culled cows, costs of drugs and veterinary services, andincreased labor costs. In addition to its prevalence within the bovinedairy industry, mastitis caused by gram-positive cocci is also commonamong goats and sheep. Additional animal diseases caused by S. aureusinclude botryomycosis in horses, purulent synovitis and osteomyelitis inpoultry, snuffles in rabbits, abortions in swine, and tick pyemia inlambs. Other species of staphylococci are major skin pathogens of canine(S. intermedius) and swine (S. hycius). In poultry species,staphylococcal pathogens cause endorcarditis and septicemia.

Staphylococcal Infections in Humans

Staphylococcus spp. are also human pathogens causing a wide variety ofinfections. The species Staphylococcus aureus, a common colonizer ofhuman mucosa and skin, is an opportunistic pathogen that can causediverse human infections. For example, S. aureus is the causative agentof several skin infections, including impetigo, furunculosis,cellulitus, and scalded skin syndrome, as well as potentially fatalpost-surgical wound infections. In addition, the exposure ofimmunocompromised individuals to S. aureus in hospital settings hasresulted in organ infections such as pneumonia, urinary tractinfections, osteomyelitis, arthritis, bacteremia, and endocarditis. S.aureus is also the causative agent of toxinoses, most notably toxicshock syndrome and food poisoning. Food poisoning caused by thestaphylococcal enterotoxin B is the most common cause of food-borneillness, surpassing even salmonellosis, campylobacteriosis andlisteriosis. Other species of staphylococci also cause human disease; S.epidermidis, S. haemolyticus and S. hominis commonly infect implantedmedical devices and S. saprophyticus is associated with urinary tractinfections in women.

Virulence Mechanisms of Staphylococci

Staphylococci infect a variety of host tissues and evade the immunesystem through the production of several types of secreted proteins,surface expressed virulence factors and metabolic systems designed forsurvival amidst the limited resources and active defenses associatedwith the host environment. Colonization is the necessary first step inestablishing infection; numerous factors including capsule, lipoteichoicacid, and teichoic acid are common structural components contributing tocolonization. In addition, surface proteins such as staphylococcalfibronectin-binding protein and bone-sialoprotein binding proteinsspecifically bind host tissue components. Toxins are commonly producedamong staphylococcal pathogens and are highly damaging; several humandiseases, including food poisoning, toxic shock syndrome and exfoliativeskin conditions, are the direct result of extracellular secreted toxinproteins. A single isolate may encode genes for 20-30 different secretedtoxins. Some of the secreted protein products are superantigens that canbind nonspecifically to the MHC class II molecule of anantigen-presenting cell and, simultaneously, to the T-cell receptor of aT cell. The binding induces T cell signaling and leads to the release ofhigh levels of proinflammatory factors, ultimately inducing host damagedue to the overwhelming immune response. Another class of virulencefactors expressed on the surface disguise the bacteria from the hostimmune system. For example, the S. aureus surface-expressed Protein Ainhibits opsonization and phagocytosis by binding of the Fc component ofhost antibody. Numerous proteases, hemolysins (alpha, beta, gamma anddelta), nucleases, lipases, hyaluronidase, and collagenase also aidbacteria in extracting nutrients from surrounding cells and protectingthem against host defenses.

Antibiotic Resistance Among Staphylococci

The CDC estimates that each year nearly 2 million people in the UnitedStates acquire a nosocomial infection, resulting in 90,000 deathsannually. Of these fatal infections, 70% are caused byantibiotic-resistant bacteria. The increase in antibiotic-resistanceamong microbial species is particularly pronounced in skin and mucosalcolonizers such as S. aureus. For example, the vast majority of S.aureus isolated from hospital settings are resistant to penicillin, and50% are also resistant to the semisynthetic penicillins, such asmethicillin, nafcillin, and oxacillin. These isolates, referred to asMRSA (methicillin resistant S. aureus) were first seen in the 1970s, andare now firmly established in hospital settings. Recently there havebeen several cases of MRSA infections in the community, where theinfected individuals had no previous exposure to hospitals or healthcareworkers. This alarming trend is intensified by the isolation of MRSAisolates that are less susceptible to vancomycin, a glycopeptide used totreat MRSA. Very few strains have been shown to be truly resistant tovancomycin according to the CDC's definition of vancomycin resistance,but several MRSA strains have been characterized as consisting ofsubpopulations with reduced susceptibility to vancomycin, or VISA(vancomycin intermediate S. aureus). Since the isolation of vancomycinresistant and vancomycin intermediate strains is a relatively newdevelopment, there is little data concerning their prevalence inhospitals and/or the community. Occasionally, VRSA (vancomycin resistantS. aureus) with full resistance to vancomycin and carrying a resistanceplasmid likely acquired from Enterococcus spp. have also been recoveredfrom humans.

Strategies for the Prevention and Treatment of Staphylococcus Infections

The emergence of numerous gram-positive pathogens that are resistant tomultiple antibiotics has fueled research efforts aimed at developingpreventative vaccines to protect against disease. Vaccines are designedto be administered to patients in order to elicit a long-term memoryresponse from the immune system, so that if the pathogen is encounteredat a future time, the immune system can more quickly and efficientlyclear the pathogen. To date, a broadly-protective vaccine againstgram-positive pathogens associated with a number of severe humandiseases, particularly those disease associated with staphylococcalinfections, is not available. Vaccine development approaches for theprevention of staphylococcal infections include those reporting the useof microbial surface components recognizing adhesion matrix molecules[MSCRAMMS (Nilsson et al. 1998. J Clin Invest 101:2640-9; Menzies et al.2002. J Infect Dis 185:937-43; Fattom et al. 2004. Vaccine 22:880-7],surface polysaccharides (McKenney et al. 2000; McKenney et al. 1999.Science 284:1523-7; Maira-Litran et al. 2002. Infect Immun 70:4433-40;Maira-Litran et al. 2004. Vaccine 22:872-9; Maira-Litran et al. 2005.Infect Immun 73:6752-62) and mutated exoproteins (Lowell et al. 1996.Infect Immun 64:4686-93; Stiles et al. 2001. Infect Immun 69:2031-6;Gampfer et al. 2002. Vaccine 20:3675-84), as antigens in subunit vaccinecompositions, as well as one live avirulent strain (Reinoso et al. 2002.Can J Vet Res 66:285-8) and several DNA vaccine approaches (Ohwada etal. 1999. J Antimicrob Chemother 44:767-74); Brouillette et al. 2002.Vaccine 20:2348-57; Senna et al. 2003. Vaccine 21:2661-6). Although manyof these compositions have shown some degree of protection, they haveachieved little cross-protection against diverse staphyloccocal strainsand have additionally failed to elicit substantial immune responses inimmunocompromised patients, an important at-risk population fornosocomial infections.

The most severe staphylococcal diseases are those mediated by theaforementioned supernantigenic pyrogenic exotoxins (SPEs) thatnonspecifically stimulate T-cells independent of antigen presentation.Such diseases include toxic shock syndrome, exfoliative skin disease,and possibly Kawasaki syndrome. For these SPE-mediated diseases,immunotherapeutic agents that boost the immune system during an activeinfection are often more effective than vaccines, which are typicallyadministered prior to infection. The overwhelming nature of the immuneresponse to SPE necessitates rapid reduction in toxin activity as thefirst objective in therapy. To date, toxin neutralization in S.aureus-mediated disease has been most effectively accomplished by theadministration of intravenous human immunoglobulin (IVIG), a purified,concentrated human antibody preparation from several thousand humandonors (Takei et al. 1993. J Clin Invest 91:602-7; Stohl and Elliot.1996. Clin Immunol Immunopathol 79:122-33). The widespread distributionof S. aureus, which colonizes approximately 30% of healthy human adults,coincides with high exposure rates for the majority of the population,so the level of anti-staphylococcal anti-toxin antibodies in IVIG isoften sufficient to neutralize toxin long enough to stabilize the immuneresponse until the bacterial load is reduced with antibiotics(Schlievert, 2001. J Allergy Clin Immunol 108(4 Suppl):S107-110). IVIGpreparations from multiple manufacturers have been shown to neutralizetoxin in proliferation assays with human peripheral blood mononuclearcells, inhibit toxin-induced human T cell-driven B cell differentiationin vitro (Stohl and Elliot. 1996. Clin Immunol Immunopathol 79:122-33;Stohl and Elliott. 1995. J Immunol 155:1838-50; Stohl et al. 1994. JImmunol 153:117-27) and reduce IL-4 and IL-2 secretion in PBMCsstimulated with staphylococcal enterotoxin B (Takei et al. 1993. J ClinInvest 91:602-7; Darenberg et al. 2004. Clin Infect Dis 38:836-42). IVIGtherapy, with its proven ability to neutralize SPE, is now a recommendedtherapy for Kawasaki syndrome and is gaining favor as a treatment methodfor staphylococcal toxic shock syndrome (Schlievert 2001. J Allergy ClinImmunol 108(4 Suppl):S107-110). Use of IVIG as an immunoprotective woundlavage during surgery has also been investigated in mice (Poelstra etal. 2000. Tissue Eng 6(4):401-411). Although standard IVIG has utilityfor limiting the advance of some staphylococcal SPE-mediated disease,the safety, efficacy and consistency of human IVIG preparationsgenerated from thousands of unselected human donors remainscontroversial (Baker et al. 1992. N Engl J Med 327:213-9; Miller et al.2001. J Allergy Clin Immunol 108:S91-4; Sacher, 2001. J Allergy ClinImmunol 108:S139-46; Darenberg et al. 2004. Clin Infect Dis 38:836-42).Furthermore, the benefit of IVIG in preventing some staphylococcalinfections is doubtful (Baker et al. 1992. N Engl J Med 327:213-9; Hill,H. R. 2000. J Pediatr 137:595-7; Darenberg et al. 2004. Clin Infect Dis38:836-42). In order to increase the effectiveness of IVIG in treatingstaphylococcal infections in certain at-risk populations, aplasma-derived, donor-selected, polyclonal anti-staphylococcal human IgGwith high titers of antibody directed toward the staphylococcal MSCRAMMSclumping factor A (ClfA) and fibrinogen-binding protein G (SdrG) wascreated and tested with success in very low birthweight infants toprevent staphylococcal sepsis (Vernachio et al. 2003. Antimicrob AgentsChemother 47:3400-6; Bloom et al. 2005. Pediatr Infect Dis J 24:858-866;Capparelli et al. 2005. Antimicrob Agents Chemother 49:4121-7). Aspecific humanized monoclonal antibody toward the S. aureus MSCRAMMClumping factor A, is also being developed. The antibody was selectedfrom a pool of thousands of murine anti-ClfA antibodies for its abilityto bind ClfA in a manner that abrogates S. aureus binding to humanfibronectin and was subsequently humanized by mutating specific targetedresidues to mimic the homologous human germline subgroup antibody (Hallet al. 2003. Infect Immun 71:6864-70; Domanski et al. 2005. Infect Immun73:5229-32). The specific antibody is being designed for use inconjunction with antibiotics for the treatment of severelife-threatening S. aureus infection, although animal studies alsodemonstrated a prophylactic protective effect.

SUMMARY

In one aspect, the present invention provides compositions including twoor more isolated polypeptides. An isolated polypeptide in thecomposition have a molecular weight as determined by electrophoresis ona sodium dodecyl sulfate-polyacrylamide gel of 88 kDa, 55 kDa, 38 kDa,37 kDa, 36 kDa, 35 kDa, or 33 kDa. For instance, a composition mayinclude isolated proteins of 88 kDa and 55 kDa. In some aspects thecomposition may include isolated polypeptides having molecular weightsof 88 kDa, 55 kDa, 38 kDa, 37 kDa, 36 kDa, 35 kDa, and 33 kDa. Thepolypeptides are isolatable from a Staphylococcus aureus when incubatedin media including an iron chelator and not isolatable when grown in themedia without the iron chelator. The composition protects an animal,such as a mouse or cow or human, against challenge with an S. aureusstrain such as, for instance, ATCC strain 19636. The composition mayfurther include a pharmaceutically acceptable carrier, and may furtherinclude one or more isolated polypeptides having a molecular weight of150 kDa, 132 kDa, 120 kDa, 75 kDa, 58 kDa, 50 kDa, 44 kDa, 43 kDa, 41kDa, or 40 kDa and isolatable from a S. aureus when grown in the mediawithout the iron chelator. In some aspects the polypeptides of thecomposition may be isolated from S. aureus ATCC strain 19636.

In some embodiments, each polypeptide of the composition has a massfingerprint of at least 80% similarity to a mass fingerprint of apolypeptide of the same molecular weight polypeptide expressed byStaphylococcus aureus ATCC strain 19636, wherein the polypeptide isisolatable from a Staphylococcus aureus when incubated in mediacomprising an iron chelator and not isolatable when grown in the mediawithout the iron chelator. For instance, the isolated polypeptide with amolecular weight of 88 kDa has a mass fingerprint of at least 80%similarity to a mass fingerprint of a 88 kDa polypeptide expressed byStaphylococcus aureus ATCC strain 19636, and the isolated polypeptidewith a molecular weight of 55 kDa has a mass fingerprint of at least 80%similarity to a mass fingerprint of a 55 kDa polypeptide expressed byStaphylococcus aureus ATCC strain 19636.

In another aspect, the present invention provides compositions thatinclude an isolated polypeptide having at least 80% sequence similarityto an amino acid sequence selected from SEQ ID NO:408 and SEQ ID NO:397.The composition may further include at least one second polypeptide,wherein the second polypeptide is isolatable from a Staphylococcusaureus when incubated in media comprising an iron chelator and notisolatable when grown in the media without the iron chelator. In somecases, the second polypeptide can include an amino acid sequence havingat least 80% similarity to an amino acid sequence selected from SEQ IDNO:353, SEQ ID NO:364, SEQ ID NO:375, SEQ ID NO:386, and SEQ ID NO:419.In other cases, the second polypeptide can have a molecular weight asdetermined by electrophoresis on a sodium dodecyl sulfate-polyacrylamidegel of 88 kDa, 55 kDa, 38 kDa, 37 kDa, 36 kDa, 35 kDa, or 33 kDa. Thecomposition may further include one or more isolated polypeptidesisolatable from a S. aureus when grown in media without the ironchelator and has a molecular weight of 150 kDa, 132 kDa, 120 kDa, 75kDa, 58 kDa, 50 kDa, 44 kDa, 43 kDa, 41 kDa, 40 kDa.

The present invention also provides methods for using the compositions.In one aspect the method is for treating in infection in a subject, andincludes administering an effective amount of a composition of thepresent invention to a subject having or at risk of having an infectioncaused by a Staphylococcus spp. In another aspect, the method is fortreating a symptom in a subject, and it includes administering aneffective amount of a composition of the present invention to a subjecthaving an infection caused by a Staphylococcus spp. The subject may be amammal, such as a human, horse, or cow. The Staphylococcus spp. may beS. aureus.

The present invention further provides methods for using antibody, forinstance, polyclonal antibody, that specifically binds polypeptides ofthe present invention. In one aspect, the method is for treating aninfection in a subject, and includes administering an effective amountof a composition to a subject having or at risk of having an infectioncaused by a Staphylococcus spp., wherein the composition includesantibody that specifically binds at least one, and in some cases morethan one, isolated polypeptide of the present invention. In anotheraspect, the method is for treating a symptom in a subject, and includesadministering an effective amount of a composition to a subject havingan infection caused by a Staphylococcus spp., wherein the compositionincludes antibody that specifically binds at least one, and in somecases more than one, isolated polypeptide of the present invention. Thesubject may be a mammal, such as a human, horse, or cow. TheStaphylococcus spp. may be S. aureus.

Also provided by the present invention are methods for decreasingcolonization in a subject. In one aspect, the method includesadministering an effective amount of a composition of the presentinvention to a subject colonized by a Staphylococcus spp. In anotheraspect, the method includes administering an effective amount of acomposition to a subject colonized by Staphylococcus spp., wherein thecomposition includes antibody that specifically binds at least one, andin some cases more than one, isolated polypeptide of the presentinvention.

The present invention provides a kit for detecting antibody thatspecifically binds a polypeptide. The kit includes, in separatecontainers, an isolated polypeptide of the present invention, and areagent that detects an antibody that specifically binds thepolypeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The electrophoretic profile of the proteins of different strainsStaphylococcus aureus derived from different species grown with andwithout iron (lanes marked Fe⁺⁺ and DP, respectively).

FIG. 2. The difference in mortality between vaccinated andnon-vaccinated mice after homologous and heterologous challenge withStaphylococcus aureus.

FIG. 3. Kaplan-Meier survival curve showing percent survival aftervaccination and homologous challenge with S. aureus ATCC 19636.

FIG. 4. Kaplan-Meier survival curve showing percent survival aftervaccination and heterologous challenge with S. aureus ATCC 19636.

FIG. 5. Kaplan-Meier survival curve showing percent survival afterpassive immunization and homologous challenge with S. aureus ATCC 19636.

FIG. 6. Kaplan-Meier survival curve showing percent survival afterpassive immunization and heterologous challenge with S. aureus strain1477.

FIG. 7. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus ATCC 19636. (SEQ ID NO:353).

FIG. 8. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:354).

FIG. 9. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:355).

FIG. 10. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:356).

FIG. 11. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:357).

FIG. 12. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:358).

FIG. 13. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:359).

FIG. 14. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:360).

FIG. 15. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:361).

FIG. 16. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:362).

FIG. 17. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:363).

FIG. 18. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus ATCC19636. (SEQ ID NO:364).

FIG. 19. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:365).

FIG. 20. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:366).

FIG. 21. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:367).

FIG. 22. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:368).

FIG. 23. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:369).

FIG. 24. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:370).

FIG. 25. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:371).

FIG. 26. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:372).

FIG. 27. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:373).

FIG. 28. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:374).

FIG. 29. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus ATCC19636. (SEQ ID NO:375).

FIG. 30. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:376).

FIG. 31. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:377).

FIG. 32. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:378).

FIG. 33. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:379).

FIG. 34. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:380).

FIG. 35. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:381).

FIG. 36. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:382).

FIG. 37. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:383).

FIG. 38. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:384).

FIG. 39. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:385).

FIG. 40. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus ATCC19636. (SEQ ID NO:386).

FIG. 41. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:387).

FIG. 42. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:388).

FIG. 43. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:389).

FIG. 44. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:390).

FIG. 45. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:391).

FIG. 46. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:392).

FIG. 47. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:393).

FIG. 48. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:394).

FIG. 49. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:395).

FIG. 50. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:396).

FIG. 51. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus ATCC19636. (SEQ ID NO:397).

FIG. 52. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:398).

FIG. 53. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:399).

FIG. 54. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:400).

FIG. 55. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:401).

FIG. 56. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:402).

FIG. 57. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:403).

FIG. 58. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:404).

FIG. 59. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:405).

FIG. 60. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:406).

FIG. 61. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:407).

FIG. 62. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus ATCC19636. (SEQ ID NO:408).

FIG. 63. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:409).

FIG. 64. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:410).

FIG. 65. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:411).

FIG. 66. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:412).

FIG. 67. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:413).

FIG. 68. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:414).

FIG. 69. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:415).

FIG. 70. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:416).

FIG. 71. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:417).

FIG. 72. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:418).

FIG. 73. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus ATCC19636. (SEQ ID NO:419).

FIG. 74. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:420).

FIG. 75. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:421).

FIG. 76. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:422).

FIG. 77. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:423).

FIG. 78. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:424).

FIG. 79. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:425).

FIG. 80. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:426).

FIG. 81. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:427).

FIG. 82. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:428).

FIG. 83. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:429).

FIG. 84. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus ATCC 19636. (SEQ ID NO:430).

FIG. 85. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:431).

FIG. 86. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:432).

FIG. 87. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:433).

FIG. 88. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:434).

FIG. 89. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:435).

FIG. 90. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:436).

FIG. 91. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:437).

FIG. 92. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:438).

FIG. 93. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:439).

FIG. 94. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:440).

FIG. 95. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus ATCC19636. (SEQ ID NO:441).

FIG. 96. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:442).

FIG. 97. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:443).

FIG. 98. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:444).

FIG. 99. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:445).

FIG. 100. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:446).

FIG. 101. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:447).

FIG. 102. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:448).

FIG. 103. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:449).

FIG. 104. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:450).

FIG. 105. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:451).

FIG. 106. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus ATCC19636. (SEQ ID NO:452).

FIG. 107. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:453).

FIG. 108. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:454).

FIG. 109. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:455).

FIG. 110. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:456).

FIG. 111. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:457).

FIG. 112. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:458).

FIG. 113. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:459).

FIG. 114. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:460).

FIG. 115. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:461).

FIG. 116. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:462).

FIG. 117. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus ATCC19636. (SEQ ID NO:463).

FIG. 118. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:464).

FIG. 119. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:465).

FIG. 120. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:466).

FIG. 121. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:467).

FIG. 122. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:468).

FIG. 123. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:469).

FIG. 124. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:470).

FIG. 125. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:471).

FIG. 126. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:472).

FIG. 127. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:473).

FIG. 128. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus ATCC19636. (SEQ ID NO:474).

FIG. 129. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:475).

FIG. 130. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:476).

FIG. 131. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:477).

FIG. 132. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:478).

FIG. 133. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:479).

FIG. 134. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:480).

FIG. 135. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:481).

FIG. 136. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:482).

FIG. 137. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:483).

FIG. 138. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:484).

FIG. 139. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus ATCC19636. (SEQ ID NO:485).

FIG. 140. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:486).

FIG. 141. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:487).

FIG. 142. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:488).

FIG. 143. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:489).

FIG. 144. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:490).

FIG. 145. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:491).

FIG. 146. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:492).

FIG. 147. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:493).

FIG. 148. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:494).

FIG. 149. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:495).

FIG. 150. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus ATCC19636. (SEQ ID NO:496).

FIG. 151. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:497).

FIG. 152. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:498).

FIG. 153. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:499).

FIG. 154. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:500).

FIG. 155. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:501).

FIG. 156. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:502).

FIG. 157. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:503).

FIG. 158. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:504).

FIG. 159. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:505).

FIG. 160. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:506).

FIG. 161. Kaplan-Meier survival curves showing percent survival afterpassive immunization and homologous challenge with S. aureus ATCC 25904.A, intravenous challenge after vaccination with rMntC; B,intraperitoneal challenge after 2× vaccination with SIRP extract, 2×vaccination with rSIRP7, or 3× vaccination with rSIRP7; C, intravenouschallenge after vaccination with rSIRP7.

FIG. 162. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:543).

FIG. 163. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:544).

FIG. 164. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:545).

FIG. 165. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:546).

FIG. 166. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:547).

FIG. 167. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:548).

FIG. 168. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:549).

FIG. 169. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:550).

FIG. 170. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:551).

FIG. 171. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:552).

FIG. 172. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:553).

FIG. 173. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:554).

FIG. 174. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:555).

FIG. 175. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:556).

FIG. 176. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:557).

FIG. 177. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:558).

FIG. 178. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:559).

FIG. 179. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:560).

FIG. 180. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:561).

FIG. 181. The amino acid sequence of a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:562).

FIG. 182. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:563).

FIG. 183. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:564).

FIG. 184. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:565).

FIG. 185. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:566).

FIG. 186. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:567).

FIG. 187. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:568).

FIG. 188. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:569).

FIG. 189. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:570).

FIG. 190. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:571).

FIG. 191. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:572).

FIG. 192. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus RF122. (SEQ ID NO:573).

FIG. 193. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Mu50. (SEQ ID NO:574).

FIG. 194. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MRSA252. (SEQ ID NO:575).

FIG. 195. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MW2. (SEQ ID NO:576).

FIG. 196. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus Newman. (SEQ ID NO:577).

FIG. 197. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus JH9. (SEQ ID NO:578).

FIG. 198. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus USA300. (SEQ ID NO:579).

FIG. 199. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus COL. (SEQ ID NO:580).

FIG. 200. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus NCTC8325. (SEQ ID NO:581).

FIG. 201. A nucleic acid sequence encoding a metal-regulated polypeptideobtained from S. aureus MSSA476. (SEQ ID NO:582).

FIG. 202. A Western blot showing binding of mouse convalescent sera torecombinantly-produced metal-regulated polypeptides.

FIG. 203. A Western blot showing binding of sera from healthy humans torecombinantly-produced metal-regulated polypeptides.

FIG. 204. A Western blot showing binding of sera from convalescenthumans to recombinantly-produced metal-regulated polypeptides.

FIG. 205. Flow cytometry data showing S. aureus DU5875 surfaceexpression of metal-regualted polypeptides.

FIG. 206. Cytokine induction follwing vaccination with rSIRP7 andrestimulation with either SIRPextract or rSIRP7.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides polypeptides and compositions includingpolypeptides. As used herein, “polypeptide” refers to a polymer of aminoacids linked by peptide bonds. Thus, for example, the terms peptide,oligopeptide, protein, and enzyme are included within the definition ofpolypeptide. This term also includes post-expression modifications ofthe polypeptide, such as glycosylations, acetylations, phosphorylations,and the like. The term polypeptide does not connote a specific length ofa polymer of amino acids. A polypeptide may be isolatable directly froma natural source, or can be prepared with the aid of recombinant,enzymatic, or chemical techniques. In the case of a polypeptide that isnaturally occurring, such a polypeptide is typically isolated.

An “isolated” polypeptide is one that has been removed from its naturalenvironment. For instance, an isolated polypeptide is a polypeptide thathas been removed from the cytoplasm or from the membrane of a cell, andmany of the polypeptides, nucleic acids, and other cellular material ofits natural environment are no longer present.

A polypeptide characterized as “isolatable” from a particular source isa polypeptide that, under appropriate conditions, is produced by theidentified source, although the polypeptide may be obtained fromalternate sources using, for example, recombinant, chemical, orenzymatic techniques well know to those skilled in the art. Thus,characterizing a polypeptide as “isolatable” from a particular sourcedoes not imply any specific source from which the polypeptide must beobtained or any particular conditions or processes under which thepolypeptide must be obtained.

A “purified” polypeptide is one that is at least 60% free, preferably atleast 75% free, and most preferably at least 90% free from othercomponents with which they are naturally associated. Polypeptides thatare produced outside the organism in which they naturally occur, e.g.,through chemical or recombinant means, are considered to be isolated andpurified by definition, since they were never present in a naturalenvironment.

As used herein, a “polypeptide fragment” refers to a portion of apolypeptide that results from digestion of a polypeptide with aprotease.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one. The terms“comprises” and variations thereof do not have a limiting meaning wherethese terms appear in the description and claims.

A polypeptide of the present invention may be characterized by molecularweight, mass fingerprint, amino acid sequence, nucleic acid that encodesthe polypeptide, immunological activity, or any combination of two ormore such characteristics. The molecular weight of a polypeptide,typically expressed in kilodaltons (kDa), can be determined usingroutine methods including, for instance, gel filtration, gelelectrophoresis including sodium dodecyl sulfate (SDS) polyacrylamidegel electrophoresis (PAGE), capillary electrophoresis, massspectrometry, liquid chromatography (including HPLC), and calculatingthe molecular weight from an observed or predicted amino acid sequence.Unless indicated otherwise, molecular weight refers to molecular weightas determined by resolving a polypeptide using an SDS polyacrylamide gelhaving a stacking gel of about 4% and a resolving gel of about 10% underreducing and denaturing conditions.

As used herein, a “mass fingerprint” refers to a population ofpolypeptide fragments obtained from a polypeptide after digestion with aprotease. Typically, the polypeptide fragments resulting from adigestion are analyzed using a mass spectrometric method. Eachpolypeptide fragment is characterized by a mass, or by a mass (m) tocharge (z) ratio, which is referred to as an “m/z ratio” or an “m/zvalue.” Methods for generating a mass fingerprint of a polypeptide areroutine. An example of such a method is disclosed in Example 13.

A polypeptide of the present invention may be a metal-regulatedpolypeptide. As used herein, a “metal-regulated polypeptide” is apolypeptide that is expressed by a microbe at a greater level when themicrobe is grown in low metal conditions compared to growth of the samemicrobe in high metal conditions. Low metal and high metal conditionsare described herein. For instance, one class of metal-regulatedpolypeptide produced by Staphylococcus spp. is not expressed atdetectable levels during growth of the microbe in high metal conditionsbut is expressed at detectable levels during growth in low metalconditions.

Examples of metal-regulated polypeptides isolatable from S. aureus aftergrowth in low iron conditions have molecular weights of 88 kDa, 55 kDa,38 kDa, 37 kDa, 36 kDa, 35 kDa, and 33 kDa. Examples of metal-regulatedpolypeptides isolatable from S. aureus after growth in low zinc or lowcopper conditions have molecular weights of 115 kDa, 88 kDa, 80 kDa, 71kDa, 69 kDa, 35 kDa, 30 kDa, 29, kDa, and 27 kDa.

Additional examples of metal-regulated polypeptides includerecombinantly-produced versions of polypeptides described herein. Arecombinantly-produced polypeptide may include the entire amino acidsequence translatable from an mRNA transcript. Alternatively, arecombinantly-produced metal-regulated polypeptide can include afragment or portion of the entire translatable amino acid sequence. Forexample, a recombinantly-produced metal-regulated polypeptide may lack acleavable sequence at either terminal of the polypeptide—e.g., acleavable signal sequence at the amino terminal of the polypeptide.

Thus, a metal-regulated polypeptide can be a polypeptide that includesthe amino acid sequence depicted in, for example, SEQ ID NO:353, SEQ IDNO:364, SEQ ID NO:375, SEQ ID NO:386, SEQ ID NO:397, SEQ ID NO:408, andSEQ ID NO:419.

The present invention also includes polypeptides that are notmetal-regulated. Such polypeptides are expressed in the presence of ametal ion such as, for example, in the presence of ferric chloride, andalso expressed when grown in low iron conditions. Examples of suchpolypeptides isolatable from S. aureus have molecular weights of 150kDa, 132 kDa, 120 kDa, 75 kDa, 58 kDa, 50 kDa, 44 kDa, 43 kDa, 41 kDa,and 40 kDa.

Whether a polypeptide is a metal-regulated polypeptide or not can bedetermined by methods useful for comparing the presence of polypeptides,including, for example, gel filtration, gel electrophoresis includingsodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),capillary electrophoresis, mass spectrometry, and liquid chromatographyincluding HPLC. Separate cultures of a microbe are grown under highmetal conditions and under low metal conditions, polypeptides of thepresent invention are isolated as described herein, and the polypeptidespresent in each culture are resolved and compared. Typically, an equalamount of polypeptides from each culture is used. Preferably, thepolypeptides are resolved using an SDS polyacrylamide gel having astacking gel of about 4% and a resolving gel of about 10% under reducingand denaturing conditions. For instance, 30 micrograms (μg) of totalpolypeptide from each culture may be used and loaded into wells of agel. After running the gel and staining the polypeptides with CoomasieBrilliant Blue, the two lanes can be compared. When determining whethera polypeptide is or is not expressed at a detectable level, 30 μg oftotal polypeptide from a culture is resolved on an SDS-PAGE gel andstained with Coomasie Brilliant Blue using methods known in the art. Apolypeptide that can be visualized by eye is considered to be expressedat a detectable level, while a polypeptide that cannot be visualized byeye is considered to not be expressed at a detectable level.

Alternatively, whether a polypeptide is metal-regulated or not can bedetermined using microarray-based gene expression analysis. Separatecultures of a microbe are grown under high metal conditions and underlow metal conditions, RNA is extracted from cells of each culture, anddifferences in RNA expression in cells grown in high metal conditionsversus RNA expression in cells grown in low metal conditions aredetected and compared. For example, labeled cDNA can be prepared from8-10 μg of bacterial RNA using established protocols. The labeled cDNAcan be applied to a microarray of the S. aureus genome. Such microarraysare commercially available and gene expression using such arrays isroutine.

Polypeptides of the present invention may have immunological activity.“Immunological activity” refers to the ability of a polypeptide toelicit an immunological response in an animal. An immunological responseto a polypeptide is the development in an animal of a cellular and/orantibody-mediated immune response to the polypeptide. Usually, animmunological response includes but is not limited to one or more of thefollowing effects: the production of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells, directed to anepitope or epitopes of the polypeptide. “Epitope” refers to the site onan antigen to which specific B cells and/or T cells respond so thatantibody is produced. The immunological activity may be protective.“Protective immunological activity” refers to the ability of apolypeptide to elicit an immunological response in an animal thatprevents or inhibits infection by Staphylococcus spp., for instance, S.aureus. Whether a polypeptide has protective immunological activity canbe determined by methods known in the art such as, for example, methodsdescribed in Examples 5, 9, or 12. For example, a polypeptide of thepresent invention, or combination of polypeptides of the presentinvention, protects a rodent such as a mouse against challenge with aStaphylococcus spp. A polypeptide of the present invention may haveseroactive activity. “Seroactive activity” refers to the ability of acandidate polypeptide to react with antibody present in convalescentserum from an animal infected with a Staphylococcus spp., for instance,S. aureus. In some aspects, the convalescent serum may be from an animalinfected with the ATCC isolate 19636, strain SAAV1, strain 2176, orstrain 1477. Polypeptides of the present invention may haveimmunoregulatory activity. “Immunoregulatory activity” refers to theability of a polypeptide to act in a nonspecific manner to enhance animmune response to a particular antigen. Methods for determining whethera polypeptide has immunoregulatory activity are known in the art.

A polypeptide of the present invention may have the characteristics of apolypeptide expressed by a reference microbe—i.e., a referencepolypeptide. The characteristics can include, for example, molecularweight, mass fingerprint, amino acid sequence, or any combinationthereof. The reference microbe can be a gram positive, preferably amember of the family Micrococcaceae, preferably, Staphylococcus spp.,more preferably, Staphylococcus aureus. Preferred examples of strainsare detailed in Table 1.

TABLE 1 Bacterial strains. Bacterial cell Laboratory designation S.aureus ATCC isolate 19636 S. aureus strain SAAV1 S. aureus strain 1477S. aureus strain 2176

When the reference microbe is S. aureus ATCC isolate 19636, a candidatepolypeptide can be considered to be a polypeptide of the presentinvention if it has a molecular weight of 88 kDa, 55 kDa, 38 kDa, 37kDa, 36 kDa 35 kDa, or 33 kDa, and has a mass fingerprint that issimilar to the mass fingerprint of a metal-regulated polypeptideexpressed by a reference microbe and having a molecular weight of 88kDa, 55 kDa, 38 kDa, 37 kDa, 36 kDa 35 kDa, or 33 kDa, respectively.Preferably, such polypeptides are metal-regulated. For instance, acandidate polypeptide can be a polypeptide of the present invention ifit has a molecular weight of 88 kDa and has a mass fingerprint similarto the mass fingerprint of an 88 kDa metal-regulated polypeptideproduced by the reference strain S. aureus ATCC isolate 19636.

Alternatively, when the reference microbe is S. aureus ATCC isolate19636, a candidate polypeptide can be considered to be a polypeptide ofthe present invention if it has an amino acid sequence that isstructurally similar, as described in detail below, to the amino acidsequence of SEQ ID NO:353, SEQ ID NO:364, SEQ ID NO:375, SEQ ID NO:386,SEQ ID NO:397, SEQ ID NO:408, or SEQ ID NO:419.

Alternatively, when the reference microbe is S. aureus RF122, acandidate polypeptide can be considered to be a polypeptide of thepresent invention if it has an amino acid sequence that is structurallysimilar, as described in detail below, to the amino acid sequence of SEQID NO:354, SEQ ID NO:365, SEQ ID NO:376, SEQ ID NO:387, SEQ ID NO:398,SEQ ID NO:409, or SEQ ID NO:420.

Alternatively, when the reference microbe is S. aureus Mu50, a candidatepolypeptide can be considered to be a polypeptide of the presentinvention if it has an amino acid sequence that is structurally similar,as described in detail below, to the amino acid sequence of SEQ IDNO:355, SEQ ID NO:366, SEQ ID NO:377, SEQ ID NO:388, SEQ ID NO:399, SEQID NO:410, or SEQ ID NO:421.

Alternatively, when the reference microbe is S. aureus MRSA252, acandidate polypeptide can be considered to be a polypeptide of thepresent invention if it has an amino acid sequence that is structurallysimilar, as described in detail below, to the amino acid sequence of SEQID NO:356, SEQ ID NO:367, SEQ ID NO:378, SEQ ID NO:389, SEQ ID NO:400,SEQ ID NO:411, or SEQ ID NO:422.

Alternatively, when the reference microbe is S. aureus MW2, a candidatepolypeptide can be considered to be a polypeptide of the presentinvention if it has an amino acid sequence that is structurally similar,as described in detail below, to the amino acid sequence of SEQ IDNO:357, SEQ ID NO:368, SEQ ID NO:379, SEQ ID NO:390, SEQ ID NO:401, SEQID NO:412, or SEQ ID NO:423.

Alternatively, when the reference microbe is S. aureus Newman, acandidate polypeptide can be considered to be a polypeptide of thepresent invention if it has an amino acid sequence that is structurallysimilar, as described in detail below, to the amino acid sequence of SEQID NO:358, SEQ ID NO:369, SEQ ID NO:380, SEQ ID NO:391, SEQ ID NO:402,SEQ ID NO:413, or SEQ ID NO:424.

Alternatively, when the reference microbe is S. aureus JH9, a candidatepolypeptide can be considered to be a polypeptide of the presentinvention if it has an amino acid sequence that is structurally similar,as described in detail below, to the amino acid sequence of SEQ IDNO:359, SEQ ID NO:370, SEQ ID NO:381, SEQ ID NO:392, SEQ ID NO:403, SEQID NO:414, or SEQ ID NO:425.

Alternatively, when the reference microbe is S. aureus USA300, acandidate polypeptide can be considered to be a polypeptide of thepresent invention if it has an amino acid sequence that is structurallysimilar, as described in detail below, to the amino acid sequence of SEQID NO:360, SEQ ID NO:371, SEQ ID NO:382, SEQ ID NO:393, SEQ ID NO:404,SEQ ID NO:415, or SEQ ID NO:426.

Alternatively, when the reference microbe is S. aureus COL, a candidatepolypeptide can be considered to be a polypeptide of the presentinvention if it has an amino acid sequence that is structurally similar,as described in detail below, to the amino acid sequence of SEQ IDNO:361, SEQ ID NO:372, SEQ ID NO:383, SEQ ID NO:394, SEQ ID NO:405, SEQID NO:416, or SEQ ID NO:427.

Alternatively, when the reference microbe is S. aureus NCTC 8325, acandidate polypeptide can be considered to be a polypeptide of thepresent invention if it has an amino acid sequence that is structurallysimilar, as described in detail below, to the amino acid sequence of SEQID NO:362, SEQ ID NO:373, SEQ ID NO:384, SEQ ID NO:395, SEQ ID NO:406,SEQ ID NO:417, or SEQ ID NO:428.

Alternatively, when the reference microbe is S. aureus MSSA476, acandidate polypeptide can be considered to be a polypeptide of thepresent invention if it has an amino acid sequence that is structurallysimilar, as described in detail below, to the amino acid sequence of SEQID NO:363, SEQ ID NO:374, SEQ ID NO:385, SEQ ID NO:396, SEQ ID NO:407,SEQ ID NO:418, or SEQ ID NO:429.

When the reference microbe is S. aureus isolate SAAV1, a candidatepolypeptide can be considered to be a polypeptide of the presentinvention if it has a molecular weight of 88 kDa, 55 kDa, 38 kDa, 37kDa, 36 kDa, 35 kDa, or 33 kDa, and has a mass fingerprint that issimilar to the mass fingerprint of a polypeptide expressed by areference microbe and having a molecular weight of 88 kDa, 55 kDa, 38kDa, 37 kDa, 36 kDa, 35 kDa, or 33 kDa, respectively. Preferably, suchpolypeptides are metal-regulated. For instance, a candidate polypeptidecan be a polypeptide of the present invention if it has a molecularweight of 88 kDa and has a mass fingerprint similar to the massfingerprint of an 88 kDa metal-regulated polypeptide produced by thereference strain S. aureus isolate SAAV1.

When the reference microbe is S. aureus strain 2176, a candidatepolypeptide can be considered to be a polypeptide of the presentinvention if it has a molecular weight of 88 kDa, 80 kDa, 65 kDa, 55kDa, 37 kDa, 36 kDa, 35 kDa, 33 kDa, or 32 kDa, and has a massfingerprint that is similar to the mass fingerprint of a polypeptideexpressed by a reference microbe and having a molecular weight of 88kDa, 80 kDa, 65 kDa, 55 kDa, 37 kDa, 36 kDa, 35 kDa, 33 kDa, or 32 kDa,respectively. Preferably, such polypeptides are metal-regulated. Forinstance, a candidate polypeptide can be a polypeptide of the presentinvention if it has a molecular weight of 88 kDa and has a massfingerprint similar to the mass fingerprint of an 88 kDa metal-regulatedpolypeptide produced by the reference strain S. aureus isolate 2176.

When the reference microbe is S. aureus strain 1477, a candidatepolypeptide can be considered to be a polypeptide of the presentinvention if it has a molecular weight of 88 kDa, 80 kDa, 65 kDa, 55kDa, 37 kDa, 36 kDa, 35 kDa, 33 kDa, or 32 kDa, and has a massfingerprint that is similar to the mass fingerprint of a polypeptideexpressed by a reference microbe and having a molecular weight of 88kDa, 80 kDa, 65 kDa, 55 kDa, 37 kDa, 36 kDa, 35 kDa, 33 kDa, or 32 kDa,respectively. Preferably, such polypeptides are metal-regulated. Forinstance, a candidate polypeptide can be a polypeptide of the presentinvention if it has a molecular weight of 88 kDa and has a massfingerprint similar to the mass fingerprint of an 88 kDa metal-regulatedpolypeptide produced by the reference strain S. aureus isolate 1477.

As used herein, a polypeptide may be “structurally similar” to areference polypeptide if the amino acid sequence of the polypeptidepossesses a specified amount of sequence similarity and/or sequenceidentity compared to the reference polypeptide. A polypeptide also maybe “structurally similar” to a reference polypeptide if the polypeptideexhibits a mass fingerprint possessing a specified amount of identitycompared to a comparable mass fingerprint of the reference polypeptide.Thus, a polypeptide may be “structurally similar” to a referencepolypeptide if, compared to the reference polypeptide, it possesses asufficient level of amino acid sequence identity, amino acid sequencesimilarity, mass fingerprint similarity, or any combination thereof.

Polypeptide Sequence Similarity and Polypeptide Sequence Identity

Structural similarity of two polypeptides can be determined by aligningthe residues of the two polypeptides (for example, a candidatepolypeptide and any appropriate reference polypeptide described herein)to optimize the number of identical amino acids along the lengths oftheir sequences; gaps in either or both sequences are permitted inmaking the alignment in order to optimize the number of identical aminoacids, although the amino acids in each sequence must nonetheless remainin their proper order. A reference polypeptide may be a polypeptidedescribed herein or any known metal-regulated polypeptide, asappropriate. A candidate polypeptide is the polypeptide being comparedto the reference polypeptide. A candidate polypeptide can be isolated,for example, from a microbe, or can be produced using recombinanttechniques, or chemically or enzymatically synthesized.

Unless modified as otherwise described herein, a pair-wise comparisonanalysis of amino acid sequences can be carried out using the BESTFITalgorithm in the GCG package (version 10.2, Madison Wis.).Alternatively, polypeptides may be compared using the Blastp program ofthe BLAST 2 search algorithm, as described by Tatiana et al., (FEMSMicrobiol Lett, 174, 247-250 (1999)), and available on the NationalCenter for Biotechnology Information (NCBI) website. The default valuesfor all BLAST 2 search parameters may be used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapx_dropoff=50, expect=10, wordsize=3, and filter on.

In the comparison of two amino acid sequences, structural similarity maybe referred to by percent “identity” or may be referred to by percent“similarity.” “Identity” refers to the presence of identical aminoacids. “Similarity” refers to the presence of not only identical aminoacids but also the presence of conservative substitutions. Aconservative substitution for an amino acid in a polypeptide of theinvention may be selected from other members of the class to which theamino acid belongs. For example, it is well-known in the art of proteinbiochemistry that an amino acid belonging to a grouping of amino acidshaving a particular size or characteristic (such as charge,hydrophobicity and hydrophilicity) can be substituted for another aminoacid without altering the activity of a protein, particularly in regionsof the protein that are not directly associated with biologicalactivity. For example, nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and tyrosine. Polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Conservative substitutions include, for example,Lys for Arg and vice versa to maintain a positive charge; Glu for Aspand vice versa to maintain a negative charge; Ser for Thr so that a free—OH is maintained; and Gln for Asn to maintain a free —NH2. Likewise,biologically active analogs of a polypeptide containing deletions oradditions of one or more contiguous or noncontiguous amino acids that donot eliminate a functional activity—such as, for example, immunologicalactivity—of the polypeptide are also contemplated.

Thus, as used herein, reference to a polypeptide of the presentinvention and/or reference to the amino acid sequence of one or more SEQID NOs can include a polypeptide with at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%amino acid sequence similarity to the reference amino acid sequence.

Alternatively, as used herein, reference to a polypeptide of the presentinvention and/or reference to the amino acid sequence of one or more SEQID NOs can include a polypeptide with at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%amino acid sequence identity to the reference amino acid sequence.

Consequently, a polypeptide of the present invention can include certainvariants including, for example, homologous polypeptides thatoriginate—biologically and/or recombinantly—from microbial species orstrains other than the microbial species or strain from which thepolypeptide was originally isolated and/or identified.

For example, a polypeptide of the invention can include a polypeptidecommonly known as formate acetyltransferase (PflB). One embodiment ofthis polypeptide is reflected in SEQ ID NO:353. Variant embodiments arereflected in SEQ ID NO:354, SEQ ID NO:355, SEQ ID NO:356, SEQ ID NO:357,SEQ ID NO:358, SEQ ID NO:359, SEQ ID NO:360, SEQ ID NO:361, SEQ IDNO:362, and SEQ ID NO:363.

As another example, a polypeptide of the invention can include apolypeptide commonly known as oligopeptide permease, peptide-bindingprotein (Opp1A). One embodiment of this polypeptide is reflected in SEQID NO:364. Variant embodiments are reflected in SEQ ID NO:365, SEQ IDNO:366, SEQ ID NO:367, SEQ ID NO:368, SEQ ID NO:369, SEQ ID NO:370, SEQID NO:371, SEQ ID NO:372, SEQ ID NO:373, and SEQ ID NO:374.

As another example, a polypeptide of the invention can include apolypeptide commonly known as siderophore compound ABC transporterbinding protein (SirA). One embodiment of this polypeptide is reflectedin SEQ ID NO:375. Variant embodiments are reflected in SEQ ID NO:376,SEQ ID NO:377, SEQ ID NO:378, SEQ ID NO:379, SEQ ID NO:380, SEQ IDNO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384, and SEQ ID NO:385.

As another example, a polypeptide of the invention can include apolypeptide sometimes referred to herein as SYN2. One embodiment of thispolypeptide is reflected in SEQ ID NO:386. Variant embodiments arereflected in SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390,SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ IDNO:395, and SEQ ID NO:396.

As another example, a polypeptide of the invention can include apolypeptide commonly known as ferric hydroxamate-binding lipoprotein(FhuD). One embodiment of this polypeptide is reflected in SEQ IDNO:397. Variant embodiments are reflected in SEQ ID NO:398, SEQ IDNO:399, SEQ ID NO:400, SEQ ID NO:401, SEQ ID NO:402, SEQ ID NO:403, SEQID NO:404, SEQ ID NO:405, SEQ ID NO:406, and SEQ ID NO:407.

As another example, a polypeptide of the invention can include apolypeptide sometimes referred to herein as SYN1. One embodiment of thispolypeptide is reflected in SEQ ID NO:408. Variant embodiments arereflected in SEQ ID NO:409, SEQ ID NO:410, SEQ ID NO:411, SEQ ID NO:412,SEQ ID NO:413, SEQ ID NO:414, SEQ ID NO:415, SEQ ID NO:416, SEQ IDNO:417, and SEQ ID NO:418.

As another example, a polypeptide of the invention can include apolypeptide commonly known as manganese transport system membraneprotein (MntC). One embodiment of this polypeptide is reflected in SEQID NO:419. Variant embodiments are reflected in SEQ ID NO:420, SEQ IDNO:421, SEQ ID NO:422, SEQ ID NO:423, SEQ ID NO:424, SEQ ID NO:425, SEQID NO:426, SEQ ID NO:427, SEQ ID NO:428, and SEQ ID NO:429.

As another example, a polypeptide of the invention can include apolypeptide commonly known as ferrichrome ABC transporter lipoprotein(SstD). Embodiments of this polypeptide are reflected in SEQ ID NO:543,SEQ ID NO:544, SEQ ID NO:545, SEQ ID NO:546, SEQ ID NO:547, SEQ IDNO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551, and SEQ ID NO:552.

As another example, a polypeptide of the invention can include apolypeptide commonly known as iron compound ABC transporter (FhuD2).Embodiments of this polypeptide are reflected in SEQ ID NO:553, SEQ IDNO:554, SEQ ID NO:555, SEQ ID NO:556, SEQ ID NO:557, SEQ ID NO:558, SEQID NO:559, SEQ ID NO:560, SEQ ID NO:561, and SEQ ID NO:562.

A polypeptide of the present invention also can be designed to provideone or more additional sequences such as, for example, the addition ofcoding sequences for added C-terminal and/or N-terminal amino acids thatmay facilitate purification by trapping on columns or use of antibodies.Such tags include, for example, histidine-rich tags that allowpurification of polypeptides on nickel columns. Such gene modificationtechniques and suitable additional sequences are well known in themolecular biology arts.

A polypeptide of the present invention also may be designed so thatcertain amino acids at the C-terminal and/or N-terminal are deleted. Forexample, one difference between the amino acid sequences of SEQ IDNO:364 and SEQ ID NO:365 is that SEQ ID NO:365 possesses an N-terminal29 amino acid addition that is not present in the amino acid sequence ofthe reference polypeptide of SEQ ID NO:364. Similar exemplary N-terminaladditions, typically varying from about 20 amino acids to about 35 aminoacids, are apparent when one compares, for example, the amino acidsequence of reference peptide SEQ ID NO:353, SEQ ID NO:364, SEQ IDNO:375, SEQ ID NO:386, SEQ ID NO:397, SEQ ID NO:408, or SEQ ID NO:419with certain variant embodiments of the respective referencepolypeptide. Other amino acids additions and/or deletions, at either theN-terminal or the C-terminal, are possible.

A “modification” of a polypeptide of the present invention includespolypeptides (or analogs thereof such as, e.g., fragments thereof) thatare chemically or enzymatically derivatized at one or more constituentamino acid. Such modifications can include, for example, side chainmodifications, backbone modifications, and N- and C-terminalmodifications such as, for example, acetylation, hydroxylation,methylation, amidation, and the attachment of carbohydrate or lipidmoieties, cofactors, and the like, and combinations thereof. Modifiedpolypeptides of the invention may retain the biological activity—suchas, for example, immunological activity—of the unmodified polypeptide ormay exhibit a reduced or increased biological activity.

The polypeptides of the present invention (including biologically activeanalogs thereof and modifications thereof) include native (naturallyoccurring), recombinant, and chemically or enzymatically synthesizedpolypeptides. For example, a polypeptide of the present invention may beprepared by isolating the polypeptide from a natural source or may beprepared recombinantly by well known methods including, for example,preparation as fusion proteins in bacteria or other host cells.

The polypeptides expressed by a reference microbe can be obtained bygrowth of the reference microbe under low metal conditions and thesubsequent isolation of a polypeptide by the processes disclosed herein.Alternatively, polypeptides expressed by a reference microbe can beobtained by identifying genes expressed at higher levels when themicrobe is grown in low metal conditions—i.e., metal-regulated genes.The metal-regulated genes can be cloned and expressed, and the expressedmetal-regulated polypeptides may be identified by the processesdescribed herein. A candidate polypeptide can be isolatable from amicrobe or identified from a microbe, preferably a gram positivemicrobe, more preferably, a member of the family Micrococcaceae,preferably, Staphylococcus spp., more preferably, Staphylococcus aureus.

Other gram positive microbes from which polypeptides can be isolatedand/or identified include Corynebacterium spp., Enterococcus spp.,Erysipelothrix spp., Kytococcus spp., and Micrococcus spp.,Mycobacterium spp., and Erysipelothrix spp. A candidate polypeptide mayalso be produced using enzymatic or chemical techniques.

Mass Fingerprint Similarity

A candidate polypeptide may be evaluated by mass spectrometric analysisto determine whether the candidate polypeptide has a mass fingerprintsimilar to one of the polypeptides expressed by a reference microbe andreferred to above by molecular weight. Typically, the candidatepolypeptide can be isolated, for instance by resolving the candidatepolypeptide by gel electrophoresis and excising the portion of the gelcontaining the candidate polypeptide. Any gel electrophoresis methodthat separates polypeptides based on differing characteristics can beused, including 1 dimensional or 2 dimensional gel electrophoresis, aswell as liquid chromatographic separation based on, for instance,hydrophobicity, pI, or size. The candidate polypeptide can befragmented, for instance by digestion with a protease. Preferably, theprotease can cleave the peptide bond on the carboxy-terminal side of theamino acid lysine and the amino acid arginine, except when the aminoacid following the lysine or the arginine is a proline. An example ofsuch a protease is trypsin. Methods for digesting a polypeptide withtrypsin are routine and known in the art. An example of such a method isdisclosed in Example 13.

Methods for the mass spectrometric analysis of polypeptides are routineand known in the art and include, but are not limited to, matrixassisted laser desorption/ionization time of flight mass spectroscopy(MALDI-TOF MS). Typically, a mixture containing the polypeptidefragments obtained from a candidate polypeptide is mixed with a matrixthat functions to transform the laser energy to the sample and produceionized, preferably monoisotopic, polypeptide fragments. Examples ofmatrices that can be used include, for instance, sinapinic acid orcyano-4-hydroxycinnamic acid. An example of a method for the analysis ofpolypeptides by MALDI-TOF MS is described in Example 13. The ionizedpolypeptide fragments are separated according to their m/z ratio, anddetected to yield a spectrum of m/z ratio versus intensity. The spectrumincludes m/z values that represent the polypeptide fragments derivedfrom the candidate polypeptide. For any given polypeptide, the amount ofeach polypeptide fragment resulting from a trypsin digestion should beequimolar. However, it is known that trypsin digestion is not always100% efficient, for instance, some sites are more efficiently cleaved.Thus, when MALDI-TOF MS is used to determine m/z values, the intensityof each m/z value is typically not identical. Generally, a spectrum hasa background level of noise present across most of the x-axis (i.e., theaxis having the values of the m/z ratios). This background level ofnoise varies depending on the running conditions and the machine used,and is easily identified by visual inspection of the spectrum. An m/zvalue is generally considered to represent a polypeptide fragment whenthe intensity is at least 2 times greater, at least 3 times greater, orat least 4 times greater than the background level of noise. Thespectrum usually includes other m/z values that are artifacts resultingfrom, for instance, incomplete digestion, over digestion, otherpolypeptides that may be present in the mixture, or the protease used todigest the polypeptide including m/z values resulting from autolysis ofthe protease. This method of digesting a polypeptide with a protease isrecognized in the art as resulting in a mass fingerprint of greatspecificity that can be used to accurately characterize the polypeptideand distinguish it from other polypeptides.

In this aspect of the invention, when a candidate polypeptide isanalyzed by mass spectroscopy, preferably both the candidate polypeptideand the polypeptide from the reference microbe are prepared and analyzedtogether, thereby decreasing any potential artifacts resulting fromdifferences in sample handling and running conditions. Preferably, allreagents used to prepare and analyze the two polypeptides are the same.For instance, the polypeptide from the reference microbe and thecandidate polypeptide are isolated under substantially the sameconditions, fragmented under substantially the same conditions, andanalyzed by MALDI-TOF MS on the same machine under substantially thesame conditions. A candidate polypeptide may be considered to be“structurally similar” to a reference polypeptide if it exhibits a massfingerprint possessing at least 80%, at least 90%, at least 95%, orsubstantially all of the m/z values present in the spectrum of thereference microbe polypeptide and above the background level of noiseare also present in the spectrum of the candidate polypeptide. (See,e.g., United States Patent Application Publication No. 2006/0233824 A1).

In another aspect, a polypeptide can be considered to be a polypeptideof the present invention if it has a molecular weight of a referencepolypeptide described in Table 2, 3, 4, or 5 and has a mass fingerprintthat includes a subpopulation including at least a specified percentageof the polypeptide fragments of the reference polypeptide as listed inTable 2, 3, 4, or 5. For instance, a polypeptide of the presentinvention includes a polypeptide of 88 kDa and a mass fingerprint thatincludes a specified percentage of polypeptide fragments having massesof HVDVR (SEQ ID NO: 1), YSYER (SEQ ID NO: 2), IIGDYRR (SEQ ID NO: 3),IFTDYRK (SEQ ID NO: 4), ELKELGQK (SEQ ID NO: 5), YAQVKPIR (SEQ ID NO:6), QMQFFGAR (SEQ ID NO: 7), SMQPFGGIR (SEQ ID NO: 8), VSGYAVNFIK (SEQID NO: 9), NHATAWQGFK (SEQ ID NO: 10), LWEQVMQLSK (SEQ ID NO: 11),SLGKEPEDQNR (SEQ ID NO: 12), DGISNTFSIVPK (SEQ ID NO: 13), AGVITGLPDAYGR(SEQ ID NO: 14), TSTFLDIYAER (SEQ ID NO: 15), SMQPFGGIRMAK (SEQ ID NO:16), THNQGVFDAYSR (SEQ ID NO: 17), KAGVITGLPDAYGR (SEQ ID NO: 18),TLLYAINGGKDEK (SEQ ID NO: 19), IEMALHDTEIVR (SEQ ID NO: 20),AGEPFAPGANPMHGR (SEQ ID NO: 21), VALYGVDFLMEEK (SEQ ID NO: 22),KTHNQGVFDAYSR (SEQ ID NO: 23), YGFDLSRPAENFK (SEQ ID NO: 24),TSSIQYENDDIMR (SEQ ID NO: 25), KAGEPFAPGANPMHGR (SEQ ID NO: 26),RVALYGVDFLMEEK (SEQ ID NO: 27), LWEQVMQLSKEER (SEQ ID NO: 28),MLETNKNHATAWQGFK (SEQ ID NO: 29), MHDFNTMSTEMSEDVIR (SEQ ID NO: 30),YGNNDDRVDDIAVDLVER (SEQ ID NO: 31), ETLIDAMEHPEEYPQLTIR (SEQ ID NO: 32),YAQVKPIRNEEGLVVDFEIEGDFPK (SEQ ID NO: 33).

The mass fingerprint of a candidate polypeptide can be determined by amass spectrometric method, for instance by MALDI-TOF MS. The massfingerprint of a candidate polypeptide will generally have additionalpolypeptide fragments and, therefore, can have additional m/z valuesother than those listed for a polypeptide in Table 2, 3, 4, or 5. Whenthe candidate polypeptide is being compared to a polypeptide in Table 2,3, 4, or 5, the candidate polypeptide can be isolatable from a microbe,preferably a gram positive microbe, more preferably, a member of thefamily Micrococcaceae, preferably, Staphylococcus spp., more preferably,Staphylococcus aureus. Other gram positive microbes includeCorynebacterium spp., Enterococcus spp., Erysipelothrix spp., Kytococcusspp., Listeria spp., Micrococcus spp., and Mycobacterium spp., andErysipelothrix spp.

A candidate polypeptide can be obtained by growth of a microbe under lowmetal conditions and the subsequent isolation of a polypeptide by theprocesses described herein. Alternatively, a candidate polypeptide canbe obtained by recombinant expression of a polynucleotide that encodesthe candidate polypeptide.

It is well known in the art that modifications of amino acids can beaccidentally introduced during sample handling, such as oxidation, andformation of carbamidomethyl derivatives. Further, these types ofmodifications alter the m/z value of a polypeptide fragment. Forinstance, if a polypeptide fragment contains a methionine that isoxidized, the m/z value will be increased by 16 relative to the samefragment that does not contain the oxidized methionine. Accordingly,those polypeptide fragments in Tables 2, 3, 4, or 5 having the notation“oxidation (M)” have an m/z value that is increased by 16 relative tothe same fragment that does not contain the oxidized methionine. It isunderstood that the polypeptide fragments of Table 2, 3, 4, or 5 can bemodified during sample handling.

Polynucleotide Sequence Similarity and Polynucleotide Sequence Identity

Polypeptides of the invention also may be identified in terms thepolynucleotide that encodes the polypeptide. Thus, the inventionincludes polynucleotides that encode a polypeptide of the invention orhybridize, under standard hybridization conditions, to a polynucleotidethat encodes a polypeptide of the invention, and the complements of suchpolynucleotide sequences.

As used herein, reference to a polynucleotide of the present inventionand/or reference to the nucleic acid sequence of one or more SEQ ID NOscan include polynucleotides having a sequence identity of at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to an identified reference polynucleotidesequence.

TABLE 2 Characteristics of polypeptides obtained from S. aureus ATCCisolate 19636. Approximate m/z value of polypeptide Polypeptidemolecular weight in fragments resulting from Predicted amino acid SEQ IDdesignation kilodaltons (kDa)¹ trypsin digest² sequence of thepolypeptide fragment NO: P23 88 625.4 HVDVR 1 717.3 YSYER 2 892.5IIGDYRR 3 942.5 IFTDYRK 4 944.5 ELKELGQK 5 974.6 YAQVKPIR 6 984.5QMQFFGAR 7 992.5 SMQPFGGIR 8 1097.6 VSGYAVNFIK 9 1159.5 NHATAWQGFK 101261.7 LWEQVMQLSK 11 1272.7 SLGKEPEDQNR 12 1277.7 DGISNTFSIVPK 13 1289.7AGVITGLPDAYGR 14 1315.7 TSTFLDIYAER 15 1322.7 SMQPFGGIRMAK 16 1394.7THNQGVFDAYSR 17 1417.8 KAGVITGLPDAYGR 18 1421.8 TLLYAINGGKDEK 19 1426.8IEMALHDTEIVR 20 1508.8 AGEPFAPGANPMHGR 21 1513.9 VALYGVDFLMEEK 22 1522.8KTHNQGVFDAYSR 23 1543.9 YGFDLSRPAENFK 24 1571.8 TSSIQYENDDIMR 25 1636.9KAGEPFAPGANPMHGR 26 1670.0 RVALYGVDFLMEEK 27 1676.0 LWEQVMQLSKEER 281876.2 MLETNKNHATAWQGFK 29 2043.1 MHDFNTMSTEMSEDVIR 30 2078.2YGNNDDRVDDIAVDLVER 31 2285.5 ETLIDAMEHPEEYPQLTIR 32 2892.9YAQVKPIRNEEGLVVDFEIEGDFPK 33 P25 55 783.6 LHSWLK 34 911.7 KLHSWLK 35937.6 TYTFHLR 36 996.6 KFDGTGPFK 37 1025.6 QAIGHMVNR 38 1063.6 KWDVSEDGK39 1185.6 IYNSIDDAFK 40 1277.6 NLEMAMYYDK 41 1324.7 ENKQLTYTTVK 421346.7 AESLLDEAGWKK 43 1381.8 TVRQAIGHMVNR 44 1394.8 TYTFHLRDDVK 451400.7 KGETNFAFTDDR 46 1419.7 FHDGTPFDADAVK 47 1422.8 NVTDINFDMPTR 481428.8 DKIYNSIDDAFK 49 1483.8 EQAEYLQAEFKK 50 1509.8 VMPAGETAFLSMKK 511547.9 FHDGTPFDADAVKK 52 1550.9 NVTDINFDMPTRK 53 1559.9 LNINGETSDKIAER54 1788.1 EILDGQEKPATQLFAK 55 1930.1 GSSSQKEQAEYLQAEFK 56 1946.0DESADFNKNDQYWGEK 57 2100.4 IAKEILDGQEKPATQLFAK 58 2239.3VSFTQSQYELPFNEMQYK 59 2493.5 EAYQPALAELAMPRPYVFVSPK + Oxidation (M) 602900.6 DIGDMNPHVYGGSMSAESMIYEPLVR + 2 Oxidation 61 (M) 2916.6DIGDMNPHVYGGSMSAESMIYEPLVR + 3 Oxidation 62 (M) P26 38 993.6 IVYVGADEK63 996.7 QALNNPVLK 64 1237.7 ETVKIENNYK 65 1272.7 ENPDVILAMDR 66 1502.0IAATKPEVIFISGR 67 1507.9 NAVVLDYGALDVMK 68 1523.9 ALPNFLESFKDDK 691559.9 LWYFAAGSTTTTIK 70 1716.0 FGGLVYDTLGFNAVDK 71 1737.0IVYVGADEKNLIGSMK 72 1844.1 FGGLVYDTLGFNAVDKK 73 1929.1GRFGGLVYDTLGFNAVDK 74 1998.2 TVMYLLVNEGELSTFGPK 75 2234.4EVNFDKIAATKPEVIFISGR 76 3143.8 VSNSNHGQNVSNEYVNKENPDVILAMDR 77 P27 37699.5 FEYIK 78 729.4 DAWPLK 79 792.5 ASVVNFR 80 852.4 VYDQLSK 81 987.5HAMGTTEIK 82 1008.5 LIDDLYEK 83 1020.5 YKDAWPLK 84 1074.5 EKEAEDLLK 851083.6 LKPDLIVASK 86 1169.5 FEYIKNDLK 87 1182.5 KTESEWTSSK 88 1184.5YDDKVAAFQK 89 1223.5 NEKVYDQLSK 90 1278.6 IAPTVSTDTVFK 91 1497.6TESEWTSSKEWK 92 1502.7 DAWPLKASVVNFR 93 1558.8 QVDNGKDIIQLTSK 94 1605.8LIDDLYEKLNIEK 95 1623.8 IVGQEPAPNLEEISK 96 1712.8 ESIPLMNADHIFVVK 971800.9 IYAGGYAGEILNDLGFK 98 1957.0 IYAGGYAGEILNDLGFKR 99 2252.0NNQVSDDLDEITWNLAGGYK 100 3383.9 RVVTLYQGATDVAVSLGVKPVGAVESWTQKPK 101 P2836 646.4 DVWAR 102 725.5 IIKPVR 103 1068.4 IGDYTSVGTR 104 1185.5KQPNLEEISK 105 1327.6 LKPDLIIADSSR 106 1343.6 VDIVDRDVWAR 107 2080.9GPYLQLDTEHLADLNPER 108 2438.1 AGLLAHPNYSYVGQFLNELGFK 109 2789.4IVVLEYSFADALAALDVKPVGIADDGK 110 P29 35 760.5 AGWAEVK 111 1012.6TVDIPKDPK 112 1107.6 KDWEETTAK 113 1204.7 VAPTVVVDYNK 114 1238.6YLEQQEMLGK 115 1244.6 LYTYGDNWGR 116 1259.7 IAVVAPTYAGGLK 117 1281.7GGEVLYQAFGLK 118 1516.8 AGWAEVKQEEIEK 119 1683.9 LGANIVAVNQQVDQSK 1201877.1 EKPDLIIVYSTDKDIK 121 1884.0 AIGQDATVSLFDEFDKK 122 2227.1VDAGTYWYNDPYTLDFMR 123 2781.4 YAGDYIVSTSEGKPTPGYESTNMWK 124 P30 33 834.5QAIEFVK 125 864.5 YIAQLEK 126 946.5 QGTPEQMR 127 962.5 QAIEFVKK 128976.5 DKFNDIPK 129 1054.5 AMITSEGAFK 130 1202.5 SNIETVHGSMK 131 1268.6HLLVETSVDKK 132 1443.6 DIFGEVYTDSIGK 133 1450.7 TIQQTFIDNDKK 134 1454.7VVTTNSILYDMAK 135 1571.7 KDIFGEVYTDSIGK 136 1593.7 QDPHAWLSLDNGIK 1371818.9 DVKPIYLNGEEGNKDK 138 1836.9 DKQDPHAWLSLDNGIK 139 1911.9QYGITPGYIWEINTEK 140 2582.3 LTDADVILYNGLNLETGNGWFEK 141 2710.2KLTDADVILYNGLNLETGNGWFEK 142 2942.4 NVGGDNVDIHSIVPVGQDPHEYEVKPK 143¹Molecular weight as determined by SDS-PAGE. ²The m/z value of apolypeptide fragment can be converted to mass by subtracting 1 from them/z value. Each mass includes a range of plus or minus 300 parts permillion (ppm), or plus or minus 1 Da.

TABLE 3 Characteristics of polypeptides obtained from S. aureus isolateSAAV1. Approximate molecular weight m/z value of polypeptide polypeptidein kilodaltons fragments resulting from Predicted amino acid sequenceSEQ ID designation (kDa)¹ trypsin digest² of the polypeptide fragmentNO: P33A 55 783.4 LHSWLK 144 911.5 KLHSWLK 145 937.5 TYTFHLR 146 996.5KFDGTGPFK 147 1025.5 QAIGHMVNR 148 1039.4 NDQYWGEK 149 1178.5GTDSLDKDSLK 150 1185.5 IYNSIDDAFK 151 1222.6 DKYTVELNLK 152 1229.5ISTLIDNVKVK 153 1346.6 AESLLDEAGWKK 154 1355.5 EQAEYLQAEFK 155 1381.6VMPAGETAFLSMK 156 1400.5 KGETNFAFTDDR 157 1419.6 FHDGTPFDADAVK 1581422.6 NVTDINFDMPTR 159 1483.6 EQAEYLQAEFKK 160 1547.7 FHDGTPFDADAVKK161 1550.6 NVTDINFDMPTRK 162 1559.7 LNINGETSDKIAER 163 1787.9EILDGQEKPATQLFAK 164 1945.8 DESADFNKNDQYWGEK 165 2239.0VSFTQSQYELPFNEMQYK 166 2354.1 QIDDEGIFIPISHGSMTVVAPK 167 2868.1DIGDMNPHVYGGSMSAESMIYEPLVR 168 P33B 55 895.4 FPYAANGR 169 904.5 ALLHASHR170 1045.5 EEGLAIKASK 171 1384.5 GEAYFVDNNSLR 172 1435.7 TIEADYVLVTVGR173 1669.8 RPNTDELGLEELGVK 174 1841.0 NAIIATGSRPIEIPNFK 175 2179.2TSISNIYAIGDIVPGLPLAHK 176 2546.2 FVEAQHSENLGVIAESVSLNFQK 177 2587.3VVGDFPIETDTIVIGAGPGGYVAAIR 178 P35 37 699.4 FEYIK 179 729.4 DAWPLK 180792.4 ASVVNFR 181 852.4 VYDQLSK 182 1008.4 LIDDLYEK 183 1020.4 YKDAWPLK184 1074.4 EKEAEDLLK 185 1083.5 LKPDLIVASK 186 1169.5 FEYIKNDLK 1871182.4 KTESEWTSSK 188 1184.4 YDDKVAAFQK 189 1278.5 IAPTVSTDTVFK 1901558.7 QVDNGKDIIQLTSK 191 1623.7 IVGQEPAPNLEEISK 192 1712.7ESIPLMNADHIFVVK 193 1800.7 IYAGGYAGEILNDLGFK 194 1956.8IYAGGYAGEILNDLGFKR 195 2251.9 NNQVSDDLDEITWNLAGGYK 196 3227.5VVTLYQGATDVAVSLGVKPVGAVESWTQKPK 197 P38 33 864.5 YIAQLEK 198 946.4QGTPEQMR 199 976.5 DKFNDIPK 200 1054.5 AMITSEGAFK 201 1146.5 FNDIPKEQR202 1268.6 HLLVETSVDKK 203 1322.5 TIQQTFIDNDK 204 1443.6 DIFGEVYTDSIGK205 1450.6 TIQQTFIDNDKK 206 1454.6 VVTTNSILYDMAK 207 1593.7QDPHAWLSLDNGIK 208 1818.9 DVKPIYLNGEEGNKDK 209 1836.8 DKQDPHAWLSLDNGIK210 1911.9 QYGITPGYIWEINTEK 211 2942.4 NVGGDNVDIHSIVPVGQDPHEYEVKPK 212¹Molecular weight as determined by SDS-PAGE. ²The m/z value of apolypeptide fragment can be converted to mass by subtracting 1 from them/z value. Each mass includes a range of plus or minus 300 parts permillion (ppm) or plus or minus 1 Da.

TABLE 4 Characteristics of polypeptides obtained from S. aureus isolate2176. Approximate molecular weight m/z value of polypeptide polypeptidein kilodaltons fragments resulting from Predicted amino acid sequence ofSEQ ID designation (kDa)¹ trypsin digest² the polypeptide fragment NO:P478 88 736.35 IIGDYR 213 814.49 IFTDYR 214 942.42 IFTDYRK 4 945.36TGNTPDGRK 215 974.40 YAQVKPIR 6 984.27 QMQFFGAR 7 992.41 SMQPFGGIR 81087.31 EQQLDVISR 216 1097.31 VSGYAVNFIK 9 1159.37 NHATAWQGFK 10 1261.37LWEQVMQLSK 11 1289.46 AGVITGLPDAYGR 14 1315.42 TSTFLDIYAER 15 1322.39LREELSEQYR 217 1394.37 THNQGVFDAYSR 17 1417.52 KAGVITGLPDAYGR 18 1426.36IEMALHDTEIVR 20 1487.39 NHATAWQGFKNGR 218 1508.42 AGEPFAPGANPMHGR 211513.52 VALYGVDFLMEEK 22 1543.43 YGFDLSRPAENFK 24 1571.50 TSSIQYENDDIMR25 1636.56 KAGEPFAPGANPMHGR 26 1859.80 DLETIVGVQTEKPFKR 219 1876.77TMATGIAGLSVAADSLSAIK 220 2042.57 MHDFNTMSTEMSEDVIR 30 2077.68YGNNDDRVDDIAVDLVER 31 2158.88 AGVITESEVQEIIDHFIMK 221 2284.90ETLIDAMEHPEEYPQLTIR 32 2575.08 FLHSLDNLGPAPEPNLTVLWSVR 222 2628.01SGAQVGPNFEGINSEVLEYDEVFK 223 2756.06 SGAQVGPNFEGINSEVLEYDEVFKK 2243262.33 VASTITSHDAGYLDKDLETIVGVQTEKPFK 225 P479 80 625.27 HVDVR 1 736.26IIGDYR 226 814.22 IFTDYR 227 942.27 IFTDYRK 4 974.26 YAQVKPIR 6 984.18QMQFFGAR 7 992.23 SMQPFGGIR 8 1087.16 EQQLDVISR 228 1097.24 VSGYAVNFIK 91159.12 NHATAWQGFK 10 1243.14 VDDIAVDLVER 229 1261.22 LWEQVMQLSK 111272.24 SLGKEPEDQNR 12 1277.18 DGISNTFSIVPK 13 1289.21 AGVITGLPDAYGR 141315.19 TSTFLDIYAER 15 1322.21 LREELSEQYR 230 1394.16 THNQGVFDAYSR 171417.32 KAGVITGLPDAYGR 18 1426.23 IEMALHDTEIVR 20 1487.19 NHATAWQGFKNGR231 1508.25 AGEPFAPGANPMHGR 21 1513.21 VALYGVDFLMEEK 22 1522.25KTHNQGVFDAYSR 23 1543.26 YGFDLSRPAENFK 24 1571.23 TSSIQYENDDIMR 251636.29 KAGEPFAPGANPMHGR 26 1703.43 DLETIVGVQTEKPFK 232 1751.45EAVQWLYLAYLAAIK 233 1859.53 DLETIVGVQTEKPFKR 234 1876.50TMATGIAGLSVAADSLSAIK 235 1936.37 NEEGLVVDFEIEGDFPK 236 2042.43MHDFNTMSTEMSEDVIR 30 2077.45 YGNNDDRVDDIAVDLVER 31 2158.57AGVITESEVQEIIDHFIMK 237 2284.61 ETLIDAMEHPEEYPQLTIR 32 2574.77FLHSLDNLGPAPEPNLTVLWSVR 238 2627.61 SGAQVGPNFEGINSEVLEYDEVFK 239 2755.70SGAQVGPNFEGINSEVLEYDEVFKK 240 2907.65 EFIQLNYTLYEGNDSFLAGPTEATSK 2413261.91 VASTITSHDAGYLDKDLETIVGVQTEKPFK 242 3421.02TPDYNELFSGDPTWVTESIGGVGIDGRPLVTK 243 P480 65 625.35 HVDVR 1 717.38 YSYER2 733.42 LPDNFK 244 736.44 IIGDYR 245 814.33 IFTDYR 246 853.31 YGNNDDR247 942.33 IFTDYRK 4 944.39 ELKELGQK 5 974.52 YAQVKPIR 6 984.36 QMQFFGAR7 992.44 SMQPFGGIR 8 1049.44 TLLYAINGGK 248 1087.43 EQQLDVISR 2491097.51 VSGYAVNFIK 9 1159.52 NHATAWQGFK 10 1289.53 AGVITGLPDAYGR 141315.51 TSTFLDIYAER 15 1322.46 LREELSEQYR 250 1394.50 THNQGVFDAYSR 171417.65 KAGVITGLPDAYGR 18 1442.56 IEMALHDTEIVR + Oxidation (M) 2511467.60 VSGYAVNFIKLTR 252 1522.61 KTHNQGVFDAYSR 23 1524.55AGEPFAPGANPMHGR + Oxidation (M) 253 1529.64 VALYGVDFLMEEK + Oxidation(M) 254 1543.62 YGFDLSRPAENFK 24 1652.68 KAGEPFAPGANPMHGR + Oxidation(M) 255 1671.76 TSTFLDIYAERDLK 256 1766.76 VDDIAVDLVERFMTK + Oxidation(M) 257 1876.86 TMATGIAGLSVAADSLSAIK 258 2077.93 YGNNDDRVDDIAVDLVER 312225.07 DSEHTMSVLTITSNVVYGKK + Oxidation (M) 259 2575.33FLHSLDNLGPAPEPNLTVLWSVR 260 2628.25 SGAQVGPNFEGINSEVLEYDEVFK 261 2748.36NLTSMLDGYAMQCGHHLNINVFNR 262 2756.63 SGAQVGPNFEGINSEVLEYDEVFKK 2633001.02 DEKSGAQVGPNFEGINSEVLEYDEVFK 264 3420.75TPDYNELFSGDPTWVTESIGGVGIDGRPLVTK 265 P481 55 634.33 AKSNSK 266 883.24TFYPEAR 267 1014.24 QFWGHLVK 268 1131.17 WIPLMMKGR 269 1207.21VINEEFEISK 270 1324.10 NEDWQLYTAGK 271 1360.28 TLLFGPFANVGPK 272 1386.31LDRPAIESSNER 273 1565.30 IDEGTDVNFGELTR 274 1584.34 EFINPLPHISYVR 2751699.29 EIEPDWNIHVYER 276 1744.36 EPPGTPPMTVPHLDTR 277 2046.52QVTDYVFIGAGGGAIPLLQK 278 2189.43 TFYPEARNEDWQLYTAGK 279 2806.58HLGGFPISGQFLACTNPQVIEQHDAK 280 P482 37 699.28 FEYIK 281 729.26 DAWPLK282 792.33 ASVVNFR 283 852.28 VYDQLSK 284 1008.30 LIDDLYEK 285 1020.31YKDAWPLK 286 1083.43 LKPDLIVASK 287 1278.36 IAPTVSTDTVFK 288 1623.44IVGQEPAPNLEEISK 289 1712.62 ESIPLMNADHIFVVK 290 1800.61IYAGGYAGEILNDLGFK 291 1956.77 IYAGGYAGEILNDLGFKR 292 2251.77NNQVSDDLDEITWNLAGGYK 293 3227.44 VVTLYQGATDVAVSLGVKPVGAVESWTQKPK 294P483 36 646.50 DVWAR 295 672.41 KLNAVK 296 716.41 VDIVDR 297 725.61IIKPVR 298 842.50 IAPTLSLK 299 850.47 QNINSFK 300 1068.50 IGDYTSVGTR 3011075.42 MIIMTDHAK + Oxidation (M) 302 1185.53 KQPNLEEISK 303 1327.59LKPDLIIADSSR 304 1343.58 VDIVDRDVWAR 305 1592.76 LKPDLIIADSSRHK 3062081.00 GPYLQLDTEHLADLNPER 307 2438.24 AGLLAHPNYSYVGQFLNELGFK 3082789.48 IVVLEYSFADALAALDVKPVGIADDGK 309 2917.60IVVLEYSFADALAALDVKPVGIADDGKK 310 P484 35 857.38 AAAIDLAGR 311 1022.23NIEADTGMR + Oxidation (M) 312 1056.32 VVDANIAAQR 313 1075.36 ADIDLPFER314 1285.44 LVGGAGEETIIAR 315 1435.44 AMAVATEQEMKAR 316 1632.50HHTEVLENPDNISK 317 1813.65 VVEAESEVPLAMAEALR 318 1887.67VIETPFIAGVAMNGIEVK 319 2299.85 AGLALTTNQLESHYLAGGNVDR 320 2806.95TVLSKGLDSGTAFEILSIDIADVDISK 321 3337.42 AGLALTTNQLESHYLAGGNVDRVVDANIAAQR322 P485 33 625.28 ADYEK 323 864.28 YIAQLEK 324 946.23 QGTPEQMR 3251045.26 ALEQAGKSLK 326 1268.35 HLLVETSVDKK 327 1443.34 DIFGEVYTDSIGK 3281450.40 TIQQTFIDNDKK 329 1454.37 VVTTNSILYDMAK 330 1571.45KDIFGEVYTDSIGK 331 1576.44 DVKPIYLNGEEGNK 332 1593.47 QDPHAWLSLDNGIK 3331819.59 DVKPIYLNGEEGNKDK 334 1836.62 DKQDPHAWLSLDNGIK 335 1911.66QYGITPGYIWEINTEK 336 2172.83 VIAVSKDVKPIYLNGEEGNK 337 2582.00LTDADVILYNGLNLETGNGWFEK 338 2942.26 NVGGDNVDIHSIVPVGQDPHEYEVKPK 339 P48632 625.42 ADYEK 340 864.41 YIAQLEK 341 1268.48 HLLVETSVDKK 342 1443.49DIFGEVYTDSIGK 343 1450.53 TIQQTFIDNDKK 344 1454.61 VVTTNSILYDMAK 3451576.64 DVKPIYLNGEEGNK 346 1593.57 QDPHAWLSLDNGIK 347 1818.77DVKPIYLNGEEGNKDK 348 1836.78 DKQDPHAWLSLDNGIK 349 1911.81QYGITPGYIWEINTEK 350 2582.18 LTDADVILYNGLNLETGNGWFEK 351 2942.32NVGGDNVDIHSIVPVGQDPHEYEVKPK 352 ¹Molecular weight as determined bySDS-PAGE. ²The m/z value of a polypeptide fragment can be converted tomass by subtracting 1 from the m/z value. Each mass includes a range ofplus or minus 400 parts per million (ppm) or 1 Dalton.

TABLE 5 Characteristics of polypeptides obtained from S. aureus bovineisolate 1477. Approximate molecular weight m/z value of polypeptidePredicted amino acid sequence of polypeptide in kilodaltons fragmentsresulting from the polypeptide designation (kDa)¹ trypsin digest²fragment SEQ ID NO: P487 88 717.39 YSYER 2 736.52 IIGDYR 583 814.46IFTDYR 584 942.46 IFTDYRK 4 974.54 YAQVKPIR 6 984.41 QMQFFGAR 7 992.40SMQPFGGIR 8 1087.49 EQQLDVISR 585 1097.50 VSGYAVNFIK 9 1159.39NHATAWQGFK 10 1261.45 LWEQVMQLSK 11 1272.50 SLGKEPEDQNR 12 1277.50DGISNTFSIVPK 13 1289.54 AGVITGLPDAYGR 14 1315.54 TSTFLDIYAER 15 1322.53LREELSEQYR 586 1394.50 THNQGVFDAYSR 17 1417.62 KAGVITGLPDAYGR 18 1426.65IEMALHDTEIVR 20 1508.59 AGEPFAPGANPMHGR 21 1522.61 KTHNQGVFDAYSR 231543.68 YGFDLSRPAENFK 24 1877.74 TMATGIAGLSVAADSLSAIK 587 2077.86YGNNDDRVDDIAVDLVER 31 2159.08 AGVITESEVQEIIDHFIMK 588 2285.07ETLIDAMEHPEEYPQLTIR 32 2575.32 FLHSLDNLGPAPEPNLTVLWSVR 589 2628.24SGAQVGPNFEGINSEVLEYDEVFK 590 2756.41 SGAQVGPNFEGINSEVLEYDEVFKK 5913262.68 VASTITSHDAGYLDKDLETIVGVQTEKPFK 592 P488 80 625.49 HVDVR 1 814.54IFTDYR 593 942.66 IFTDYRK 4 974.69 YAQVKPIR 6 984.59 QMQFFGAR 7 992.55SMQPFGGIR 8 1159.64 NHATAWQGFK 10 1261.63 LWEQVMQLSK 11 1272.74SLGKEPEDQNR 12 1277.69 DGISNTFSIVPK 13 1289.76 AGVITGLPDAYGR 14 1315.73TSTFLDIYAER 15 1322.72 SMQPFGGIRMAK 16 1394.73 THNQGVFDAYSR 17 1417.86KAGVITGLPDAYGR 18 1422.76 TLLYAINGGKDEK 19 1426.80 IEMALHDTEIVR 201508.82 AGEPFAPGANPMHGR 21 1513.80 VALYGVDFLMEEK 22 1543.82YGFDLSRPAENFK 24 1571.82 TSSIQYENDDIMR 25 1703.99 DLETIVGVQTEKPFK 5941860.23 DLETIVGVQTEKPFKR 595 1877.07 TMATGIAGLSVAADSLSAIK 596 1937.09NEEGLVVDFEIEGDFPK 597 2078.13 YGNNDDRVDDIAVDLVER 31 2575.56FLHSLDNLGPAPEPNLTVLWSVR 598 2628.30 SGAQVGPNFEGINSEVLEYDEVFK 599 2908.63EFIQLNYTLYEGNDSFLAGPTEATSK 600 P489 65 733.67 IVKFAR 601 944.71 ELKELGQK5 974.79 YAQVKPIR 6 984.69 QMQFFGAR 7 1049.83 TLLYAINGGK 602 1087.78EQQLDVISR 603 1097.79 VSGYAVNFIK 9 1243.80 VDDIAVDLVER 604 1272.82SLGKEPEDQNR 12 1289.87 AGVITGLPDAYGR 14 1299.92 LPDNFKTYCAK 605 1315.83TSTFLDIYAER 15 1322.84 SMQPFGGIRMAK 16 1390.93 DQKGALSSLSSVAK 6061394.84 THNQGVFDAYSR 17 1577.94 VASTITSHDAGYLDK 607 1637.09KAGEPFAPGANPMHGR 26 1704.16 DLETIVGVQTEKPFK 608 2030.42MSIKTSSIQYENDDIMR 608 2078.34 YGNNDDRVDDIAVDLVER 31 2284.60ETLIDAMEHPEEYPQLTIR 32 2575.77 FLHSLDNLGPAPEPNLTVLWSVR 610 2628.64SGAQVGPNFEGINSEVLEYDEVFK 611 P490 55 883.81 TFYPEAR 612 1014.87 QFWGHLVK613 1131.97 WIPLMMKGR 614 1207.99 VINEEFEISK 615 1231.97 YSFDQVIMTK 6161325.02 NEDWQLYTAGK 617 1361.17 TLLFGPFANVGPK 618 1362.14 GREDNPGIMAASK+ Oxidation (M) 619 1387.14 LDRPAIESSNER 620 1481.24 NEDWQLYTAGKR 6211566.28 IDEGTDVNFGELTR 622 1585.34 EFINPLPHISYVR 623 1700.36EIEPDWNIHVYER 624 1761.49 EPPGTPPMTVPHLDTR + Oxidation (M) 625 2047.67QVTDYVFIGAGGGAIPLLQK 626 2208.82 VYGKEPPGTPPMTVPHLDTR + Oxidation (M)627 2865.21 HLGGFPISGQFLACTNPQVIEQHDAK 628 P492 36 857.57 AAAIDLAGR 6291056.59 VVDANIAAQR 630 1075.61 ADIDLPFER 631 1285.74 LVGGAGEETIIAR 6321632.95 HHTEVLENPDNISK 633 1814.09 VVEAESEVPLAMAEALR 634 2284.45AAAIDLAGRDVLEAVQMSVNPK + Oxidation (M) 635 2300.40AGLALTTNQLESHYLAGGNVDR 636 2807.80 TVLSKGLDSGTAFEILSIDIADVDISK 637 P49335 762.46 FVFHGR 638 964.39 DGFNNIER 639 1363.56 GHVYNGISGGQFK 6401443.56 YTPTSILYFNPK 641 1450.64 QLAEDLQKHLGAK 642 1819.88NHSEYVTDMRLIGIR + Oxidation (M) 643 1875.84 DLPPMEQVFDTLDLDK 644 1941.00IRPEDMHIMANIFLPK + Oxidation (M) 645 2081.10 RIRPEDMHIMANIFLPK 6462283.30 ISHLVLTRTGLYIIDSQLLK 647 P495 32 ¹Molecular weight as determinedby SDS-PAGE. ²The m/z value of a polypeptide fragment can be convertedto mass by subtracting 1 from the m/z value. Each mass includes a rangeof plus or minus 430 parts per million (ppm) or 1 Dalton.

In this context, “sequence identity” refers to the identity between twopolynucleotide sequences. Sequence identity is generally determined byaligning the bases of the two polynucleotides (for example, aligning thenucleotide sequence of the candidate sequence and a nucleotide sequencethat includes, for example, the nucleotide sequence of SEQ ID NO:474 orSEQ ID NO:485) to optimize the number of identical nucleotides along thelengths of their sequences; gaps in either or both sequences arepermitted in making the alignment in order to optimize the number ofshared nucleotides, although the nucleotides in each sequence mustnonetheless remain in their proper order. A candidate sequence is thesequence being compared to a known sequence—e.g., a nucleotide sequencethat includes the nucleotide sequence of, for example, SEQ ID NO:474 orSEQ ID NO:485. For example, two polynucleotide sequences can be comparedusing the Blastn program of the BLAST 2 search algorithm, as describedby Tatiana et al., FEMS Microbiol Lett., 1999; 174: 247-250, andavailable on the world wide web at ncbi.nlm.nih.gov/BLAST/. The defaultvalues for all BLAST 2 search parameters may be used, including rewardfor match=1, penalty for mismatch=−2, open gap penalty=5, extension gappenalty=2, gap x_dropoff=50, expect=10, wordsize=11, and filter on.

For example, a polynucleotide of the invention can include apolynucleotide that encodes a polypeptide commonly known as formateacetyltransferase (PflB). One embodiment of such a polynucleotide isreflected in SEQ ID NO:430. Variant embodiments are reflected in SEQ IDNO:431, SEQ ID NO:432, SEQ ID NO:433, SEQ ID NO:434, SEQ ID NO:435, SEQID NO:436, SEQ ID NO:437, SEQ ID NO:438, SEQ ID NO:439, and SEQ IDNO:440.

As another example, a polynucleotide of the invention can include apolynucleotide that encodes a polypeptide commonly known as oligopeptidepermease, peptide-binding protein (Opp1A). One embodiment of such apolynucleotide is reflected in SEQ ID NO:441. Variant embodiments arereflected in SEQ ID NO:442, SEQ ID NO:443, SEQ ID NO:444, SEQ ID NO:445,SEQ ID NO:446, SEQ ID NO:447, SEQ ID NO:448, SEQ ID NO:449, SEQ IDNO:450, and SEQ ID NO:451.

As another example, a polynucleotide of the invention can include apolynucleotide that encodes a polypeptide commonly known as siderophorecompound ABC transporter binding protein (SirA). One embodiment of sucha polynucleotide is reflected in SEQ ID NO:452. Variant embodiments arereflected in SEQ ID NO:453, SEQ ID NO:454, SEQ ID NO:455, SEQ ID NO:456,SEQ ID NO:457, SEQ ID NO:458, SEQ ID NO:459, SEQ ID NO:460, SEQ IDNO:461, and SEQ ID NO:462.

As another example, a polynucleotide of the invention can include apolynucleotide that encodes a polypeptide referred to herein as SYN2.One embodiment of such a polynucleotide is reflected in SEQ ID NO:463.Variant embodiments are reflected in SEQ ID NO:464, SEQ ID NO:465, SEQID NO:466, SEQ ID NO:467, SEQ ID NO:468, SEQ ID NO:469, SEQ ID NO:470,SEQ ID NO:471, SEQ ID NO:472, and SEQ ID NO:473.

As another example, a polynucleotide of the invention can include apolynucleotide that encodes a polypeptide commonly known as FhuD. Oneembodiment of such a polynucleotide is reflected in SEQ ID NO:474.Variant embodiments are reflected in SEQ ID NO:475, SEQ ID NO:476, SEQID NO:477, SEQ ID NO:478, SEQ ID NO:479, SEQ ID NO:480, SEQ ID NO:481,SEQ ID NO:482, SEQ ID NO:483, and SEQ ID NO:484.

As another example, a polynucleotide of the invention can include apolynucleotide that encodes a polypeptide referred to herein as SYN1.One embodiment of such a polynucleotide is reflected in SEQ ID NO:485.Variant embodiments are reflected in SEQ ID NO:486, SEQ ID NO:487, SEQID NO:488, SEQ ID NO:489, SEQ ID NO:490, SEQ ID NO:491, SEQ ID NO:492,SEQ ID NO:493, SEQ ID NO:494, and SEQ ID NO:495.

As another example, a polynucleotide of the invention can include apolynucleotide that encodes a polypeptide commonly known as MntC. Oneembodiment of such a polynucleotide is reflected in SEQ ID NO:496.Variant embodiments are reflected in SEQ ID NO:497, SEQ ID NO:498, SEQID NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:502, SEQ ID NO:503,SEQ ID NO:504, SEQ ID NO:505, and SEQ ID NO:506.

As another example, a polynucleotide of the invention can include apolynucleotide that encodes a polypeptide commonly known as ferrichromeABC transporter lipoprotein (SstD). Embodiments of such a polynucleotideare reflected in SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565, SEQ IDNO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ ID NO:570, SEQID NO:571, and SEQ ID NO:572.

As another example, a polynucleotide of the invention can include apolynucleotide that encodes a polypeptide commonly known as ironcompound ABC transporter (FhuD2). Embodiments of such a polynucleotideare reflected in SEQ ID NO:573, SEQ ID NO:574, SEQ ID NO:575, SEQ IDNO:576, SEQ ID NO:577, SEQ ID NO:578, SEQ ID NO:579, SEQ ID NO:580, SEQID NO:581, and SEQ ID NO:582.

Also provided by the present invention are whole cell preparations of amicrobe, where the microbe expresses one or more of the polypeptides ofthe present invention. The cells present in a whole cell preparation arepreferably inactivated such that the cells cannot replicate, but theimmunological activity of the polypeptides of the present inventionexpressed by the microbe is maintained. Typically, the cells are killedby exposure to agents such as glutaraldehyde, formalin, or formaldehyde.

Compositions

A composition of the present invention may include at least one isolatedpolypeptide described herein, or a number of polypeptides that is aninteger greater than one (e.g., at least two, at least three, at leastfour). For example, a composition can include an isolated polypeptidethat includes the amino acid sequence of SEQ ID NO:408 and/or anisolated polypeptide that includes the amino acid sequence of SEQ IDNO:397. Unless a specific level of sequence similarity and/or identityis expressly indicated herein (e.g., at least 80% sequence similarity,at least 90% sequence identity, etc.), reference to the amino acidsequence of an identified SEQ ID NO includes variants having the levelsof sequence similarity and/or the levels of sequence identity describedherein in the section headed “Polypeptide sequence similarity andpolypeptide sequence identity.”

In some embodiments, the composition can include one or more additionalisolated polypeptides. In some embodiments, the additional isolatedpolypeptide or polypeptides may include one or more metal-regulatedpolypeptides. Thus, a composition can include at least one isolatedmetal-regulated polypeptide that includes an amino acid sequencedepicted in one or more of SEQ ID NO:353 through SEQ ID NO:429 and/orone or more of SEQ ID NO:543 through SEQ ID NO:562. In addition or inthe alternative, a composition can include at least one isolatedmetal-regulated polypeptide having a molecular weight of 88 kDa, 55 kDa,38 kDa, 37 kDa, 36 kDa, 35 kDa, or 33 kDa. In addition or in thealternative, a composition can include at least one metal-regulatedisolated polypeptide that includes an amino acid sequence encoded by apolynucleotide that includes a nucleotide sequence depicted in one ormore of SEQ ID NO:430 through SEQ ID NO:506 and/or one or more of SEQ IDNO:563 through SEQ ID NO:582.

In one embodiment, the composition includes a polypeptide that includesthe amino acid sequence depicted in SEQ ID NO:397 (or a variant thereofsuch as, for example, any one of the amino acid sequences depicted inSEQ ID NO:398, SEQ ID NO:399, SEQ ID NO:400, SEQ ID NO:401, SEQ IDNO:402, SEQ ID NO:403, SEQ ID NO:404, SEQ ID NO:405, SEQ ID NO:406, orSEQ ID NO:407).

In another embodiment, the composition includes a polypeptide thatincludes the amino acid sequence depicted in SEQ ID NO:408 (or a variantthereof such as, for example, any one of the amino acid sequencesdepicted in SEQ ID NO:409, SEQ ID NO:410, SEQ ID NO:411, SEQ ID NO:412,SEQ ID NO:413, SEQ ID NO:414, SEQ ID NO:415, SEQ ID NO:416, SEQ IDNO:417, or SEQ ID NO:418).

In another embodiment, the composition includes a polypeptide thatincludes the amino acid sequence depicted in SEQ ID NO:419 (or a variantthereof such as, for example, any one of the amino acid sequencesdepicted in SEQ ID NO:420, SEQ ID NO:421, SEQ ID NO:422, SEQ ID NO:423,SEQ ID NO:424, SEQ ID NO:425, SEQ ID NO:426, SEQ ID NO:427, SEQ IDNO:428, or SEQ ID NO:429).

In another embodiment, the composition includes a polypeptide thatincludes the amino acid sequence depicted in SEQ ID NO:375 (or a variantthereof such as, for example, any one of the amino acid sequencesdepicted in SEQ ID NO:376, SEQ ID NO:377, SEQ ID NO:378, SEQ ID NO:379,SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ IDNO:384, or SEQ ID NO:385).

In another embodiment, the composition includes a polypeptide thatincludes the amino acid sequence depicted in SEQ ID NO:386 (or a variantthereof such as, for example, any one of the amino acid sequencesdepicted in SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390,SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ IDNO:395, or SEQ ID NO:396).

In another embodiment, the composition includes a polypeptide thatincludes the amino acid sequence depicted in SEQ ID NO:364 (or a variantthereof such as, for example, any one of the amino acid sequencesdepicted in SEQ ID NO:365, SEQ ID NO:366, SEQ ID NO:367, SEQ ID NO:368,SEQ ID NO:369, SEQ ID NO:370, SEQ ID NO:371, SEQ ID NO:372, SEQ IDNO:373, or SEQ ID NO:374).

In another embodiment, the composition includes a polypeptide thatincludes the amino acid sequence depicted in SEQ ID NO:353 (or a variantthereof such as, for example, any one of the amino acid sequencesdepicted in SEQ ID NO:354, SEQ ID NO:355, SEQ ID NO:356, SEQ ID NO:357,SEQ ID NO:358, SEQ ID NO:359, SEQ ID NO:360, SEQ ID NO:361, SEQ IDNO:362, or SEQ ID NO:363).

In another embodiment, the composition includes a polypeptide thatincludes the amino acid sequence, or a variant thereof, of any one ofthe amino acid sequences depicted in SEQ ID NO:543, SEQ ID NO:544, SEQID NO:545, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ ID NO:549,SEQ ID NO:550, SEQ ID NO:551, or through SEQ ID NO:552.

In another embodiment, the composition includes a polypeptide thatincludes the amino acid sequence, or variant thereof, of any one of theamino acid sequences depicted in SEQ ID NO:553, SEQ ID NO:554, SEQ IDNO:555, SEQ ID NO:556, SEQ ID NO:557, SEQ ID NO:558, SEQ ID NO:559, SEQID NO:560, SEQ ID NO:561, or SEQ ID NO:562.

In some embodiments, the composition can include a combination of two ormore polypeptides selected from the following: a SYN1 polypeptide, aMntC polypeptide, a FhuD polypeptide, a SYN2 polypeptide, a SirApolypeptide, an Opp1A polypeptide, and a Pflb polypeptide.

Thus, the composition can include at least one or any combination thatincludes at least two, at least three, at least four, at least five, atleast six, or all seven of: a SYN1 polypeptide, a MntC polypeptide, aFhuD polypeptide, a SYN2 polypeptide, a SirA polypeptide, an Opp1Apolypeptide, and a Pflb polypeptide. Exemplary compositions that includecombinations of polypeptides are identified in Table 6.

Composition SYN1 MntC FhuD SYN2 SirA Opp1A Pflb 2 pPeptides* 1 X X 2 X X3 X X 4 X X 5 X X 6 X X 7 X X 8 X X 9 X X 10 X X 11 X X 12 X X 13 X X 14X X 15 X X 16 X X 17 X X 18 X X 19 X X 20 X X 21 X X 3 pPeptides* 22 X XX 23 X X X 24 X X X 25 X X X 26 X X X 27 X X X 28 X X X 29 X X X 30 X XX 31 X X X 32 X X X 33 X X X 34 X X X 35 X X X 36 X X X 37 X X X 38 X XX 39 X X X 40 X X X 41 X X X 42 X X X 43 X X X 44 X X X 45 X X X 46 X XX 47 X X X 48 X X X 49 X X X 50 X X X 51 X X X 52 X X X 53 X X X 54 X XX 55 X X X 56 X X X 4 pPeptides* X X X X 57 X X X X 58 X X X X 59 X X XX 60 X X X X 61 X X X X 62 X X X X 63 X X X 64 X X X X 65 X X X X 67 X XX X 68 X X X X 69 X X X X 70 X X X X 71 X X X X 72 X X X X 73 X X X X 74X X X X 75 X X X X 76 X X X X 77 X X X X 78 X X X X 79 X X X X 80 X X XX 81 X X X X 82 X X X X 83 X X X X 84 X X X X 85 X X X X 86 X X X X 87 XX X X 88 X X X X 89 X X X X 90 X X X X 91 X X X X 5 pPeptides* 92 X X XX X 93 X X X X X 94 X X X X X 95 X X X X X 96 X X X X X 97 X X X X X 98X X X X X 99 X X X X X 100 X X X X X 101 X X X X X 102 X X X X X 103 X XX X X 104 X X X X X 105 X X X X X 106 X X X X X 107 X X X X X 108 X X XX X 109 X X X X X 110 X X X X X 111 X X X X X 112 X X X X X 6 pPeptides*113 X X X X X X 114 X X X X X X 115 X X X X X X 116 X X X X X X 117 X XX X X X 118 X X X X X X 119 X X X X X X *pPeptides = polypeptides “X”identifies polypeptides included in a particular composition.

Throughout this description, a SYN1 polypeptide may be characterized byone or more of the following: an amino acid sequence that includes theamino acid sequence depicted in any one of SEQ ID NO:408, SEQ ID NO:409,SEQ ID NO:410, SEQ ID NO:411, SEQ ID NO:412, SEQ ID NO:413, SEQ IDNO:414, SEQ ID NO:415, SEQ ID NO:416, SEQ ID NO:417, or SEQ ID NO:418,being encoded by a polynucleotide that includes the nucleic acidsequence depicted in any one of SEQ ID NO:485, SEQ ID NO:486, SEQ IDNO:487, SEQ ID NO:488, SEQ ID NO:489, SEQ ID NO:490, SEQ ID NO:491, SEQID NO:492, SEQ ID NO:493, SEQ ID NO:494, SEQ ID NO:495, and/or acalculated molecular weight of about 33.1 kDa.

Throughout this description, an MntC polypeptide may be characterized byone or more of the following: possessing a molecular weight asdetermined by SDS-PAGE of 33 kDa, a mass fingerprint at least 80%similar to the mass fingerprint of an 33 kDa metal-regulated polypeptideproduced by the reference strain S. aureus ATCC isolate 19636, an aminoacid sequence that includes the amino acid sequence depicted in any oneof SEQ ID NO:419, SEQ ID NO:420, SEQ ID NO:421, SEQ ID NO:422, SEQ IDNO:423, SEQ ID NO:424, SEQ ID NO:425, SEQ ID NO:426, SEQ ID NO:427, SEQID NO:428, or SEQ ID NO:429, being encoded by a polynucleotide thatincludes the nucleic acid sequence depicted in any one of SEQ ID NO:496,SEQ ID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQ IDNO:501, SEQ ID NO:502, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, orSEQ ID NO:506, and/or a calculated molecular weight of about 34.6 kDa.

Throughout this description, a FhuD polypeptide may be characterized byone or more of the following: an amino acid sequence that includes theamino acid sequence depicted in any one of SEQ ID NO:397, SEQ ID NO:398,SEQ ID NO:399, SEQ ID NO:400, SEQ ID NO:401, SEQ ID NO:402, SEQ IDNO:403, SEQ ID NO:404, SEQ ID NO:405, SEQ ID NO:406, or SEQ ID NO:407,being encoded by a polynucleotide that includes the nucleic acidsequence depicted in any one of SEQ ID NO:474, SEQ ID NO:475, SEQ IDNO:476, SEQ ID NO:477, SEQ ID NO:478, SEQ ID NO:479, SEQ ID NO:480, SEQID NO:481, SEQ ID NO:482, SEQ ID NO:483, or SEQ ID NO:484, and/or acalculated molecular weight of about 35.4 kDa.

Throughout this description, a SYN2 polypeptide may be characterized byone or more of the following: a molecular weight as determined bySDS-PAGE of 36 kDa, a mass fingerprint at least 80% similar to the massfingerprint of an 36 kDa metal-regulated polypeptide produced by thereference strain S. aureus ATCC isolate 19636, an amino acid sequencethat includes the amino acid sequence depicted in any one of SEQ IDNO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQID NO:391, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:395,or SEQ ID NO:396, being encoded by a polynucleotide that includes thenucleic acid sequence depicted in any one of SEQ ID NO:463, SEQ IDNO:464, SEQ ID NO:465, SEQ ID NO:466, SEQ ID NO:467, SEQ ID NO:468, SEQID NO:469, SEQ ID NO:470, SEQ ID NO:471, SEQ ID NO:472, or SEQ IDNO:473, and/or a calculated molecular weight of about 36.5 kDa.

Throughout this description, a SirA polypeptide may be characterized byone or more of the following: a molecular weight as determined bySDS-PAGE of 37 kDa, a mass fingerprint at least 80% similar to the massfingerprint of an 37 kDa metal-regulated polypeptide produced by thereference strain S. aureus ATCC isolate 19636, an amino acid sequencethat includes the amino acid sequence depicted in any one of SEQ IDNO:375, SEQ ID NO:376, SEQ ID NO:377, SEQ ID NO:378, SEQ ID NO:379, SEQID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384,or SEQ ID NO:385, being encoded by a polynucleotide that includes thenucleic acid sequence depicted in any one of SEQ ID NO:452, SEQ IDNO:453, SEQ ID NO:454, SEQ ID NO:455, SEQ ID NO:456, SEQ ID NO:457, SEQID NO:458, SEQ ID NO:459, SEQ ID NO:460, SEQ ID NO:461, or SEQ IDNO:462, and/or a calculated molecular weight of about 36.6 kDa.

Throughout this description, a Opp1A polypeptide may be characterized byone or more of the following: a molecular weight as determined bySDS-PAGE of 55 kDa, a mass fingerprint at least 80% similar to the massfingerprint of an 55 kDa metal-regulated polypeptide produced by thereference strain S. aureus ATCC isolate 19636, an amino acid sequencethat includes the amino acid sequence depicted in any one of SEQ IDNO:364, SEQ ID NO:365, SEQ ID NO:366, SEQ ID NO:367, SEQ ID NO:368, SEQID NO:369, SEQ ID NO:370, SEQ ID NO:371, SEQ ID NO:372, SEQ ID NO:373,or SEQ ID NO:374, being encoded by a polynucleotide that includes thenucleic acid sequence depicted in any one of SEQ ID NO:441, SEQ IDNO:442, SEQ ID NO:443, SEQ ID NO:444, SEQ ID NO:445, SEQ ID NO:446, SEQID NO:447, SEQ ID NO:448, SEQ ID NO:449, SEQ ID NO:450, or SEQ IDNO:451, and/or a calculated molecular weight of about 59.9 kDa.

Throughout this description, a PflB polypeptide may be characterized byone or more of the following: a molecular weight as determined bySDS-PAGE of 88 kDa, a mass fingerprint at least 80% similar to the massfingerprint of an 88 kDa metal-regulated polypeptide produced by thereference strain S. aureus ATCC isolate 19636, an amino acid sequencethat includes the amino acid sequence depicted in any one of SEQ IDNO:353, SEQ ID NO:354, SEQ ID NO:355, SEQ ID NO:356, SEQ ID NO:357, SEQID NO:358, SEQ ID NO:359, SEQ ID NO:360, SEQ ID NO:361, SEQ ID NO:362,or SEQ ID NO:363, being encoded by a polynucleotide that includes thenucleic acid sequence depicted in any one of SEQ ID NO:430, SEQ IDNO:431, SEQ ID NO:432, SEQ ID NO:433, SEQ ID NO:434, SEQ ID NO:435, SEQID NO:436, SEQ ID NO:437, SEQ ID NO:438, SEQ ID NO:439, or SEQ IDNO:440, and/or a calculated molecular weight of about 84.7 kDa.

In another particular embodiment, the composition can include acombination of polypeptides such as, for example, a MntC polypeptide, aFhuD polypeptide, a SirA polypeptide, and a SYN2 polypeptide(Composition 77 of Table 6), each polypeptide being characterized asdescribed immediately above.

In some embodiments, a composition can include one or more polypeptidesthat are produced recombinantly. For example, a composition can includea recombinantly-produced Pflb polypeptide such as, for example, apolypeptide that includes the amino acid sequence depicted in SEQ IDNO:353, although a recombinantly-produced Pflb polypeptide may becharacterized in any manner in which a Pflb polypeptide may becharacterized, as described above, in addition to beingrecombinantly-produced. Such a composition can include one or morerecombinantly-produced polypeptides, one or more polypeptides isolatedfrom S. aureus, or any combination thereof.

As another example, a composition can include a recombinantly-producedOpp1A polypeptide such as, for example, a polypeptide that includes theamino acid sequence depicted in SEQ ID NO:364, although arecombinantly-produced Opp1A polypeptide may be characterized in anymanner in which an Opp1A polypeptide may be characterized, as describedabove, in addition to being recombinantly-produced. Such a compositioncan further include one or more recombinantly-produced polypeptides, oneor more polypeptides isolated from S. aureus, or any combinationthereof.

As another example, a composition can include a recombinantly-producedSirA polypeptide such as, for example, a polypeptide that includes theamino acid sequence depicted in SEQ ID NO:375, although arecombinantly-produced SirA polypeptide may be characterized in anymanner in which a SirA polypeptide may be characterized, as describedabove, in addition to being recombinantly-produced. Such a compositioncan further include one or more recombinantly-produced polypeptides, oneor more polypeptides isolated from S. aureus, or any combinationthereof.

As another example, a composition can include a recombinantly-producedSYN2 polypeptide such as, for example, a polypeptide that includes theamino acid sequence depicted in SEQ ID NO:386, although arecombinantly-produced SYN2 polypeptide may be characterized in anymanner in which a SYN2 polypeptide may be characterized, as describedabove, in addition to being recombinantly-produced. Such a compositioncan further include one or more recombinantly-produced polypeptides, oneor more polypeptides isolated from S. aureus, or any combinationthereof.

As another example, a composition can include a recombinantly-producedFhuD polypeptide such as, for example, a polypeptide that includes theamino acid sequence depicted in SEQ ID NO:397, although arecombinantly-produced FhuD polypeptide may be characterized in anymanner in which a FhuD polypeptide may be characterized, as describedabove, in addition to being recombinantly-produced. Such a compositioncan further include one or more recombinantly-produced polypeptides, oneor more polypeptides isolated from S. aureus, or any combinationthereof.

As another example, a composition can include a recombinantly-producedSYN1 polypeptide such as, for example, a polypeptide that includes theamino acid sequence depicted in SEQ ID NO:408, although arecombinantly-produced SYN1 polypeptide may be characterized in anymanner in which a SYN1 polypeptide may be characterized, as describedabove, in addition to being recombinantly-produced. Such a compositioncan further include one or more recombinantly-produced polypeptides, oneor more polypeptides isolated from S. aureus, or any combinationthereof.

As another example, a composition can include a recombinantly-producedMntC polypeptide such as, for example, a polypeptide that includes theamino acid sequence depicted in SEQ ID NO:419, although arecombinantly-produced MntC polypeptide may be characterized in anymanner in which a MntC polypeptide may be characterized, as describedabove, in addition to being recombinantly-produced. Such a compositioncan further include one or more recombinantly-produced polypeptides, oneor more polypeptides isolated from S. aureus, or any combinationthereof.

In some embodiments, a recombinantly-produced polypeptide can representan immunologically active fragment of the native version of thepolypeptide. An immunologically active fragment may include amino acidadditions to the amino terminal and/or carboxy terminal of the core(e.g., the amino acid sequence of SEQ ID NO: 353, SEQ ID NO: 364, SEQ IDNO: 375, SEQ ID NO: 386, SEQ ID NO: 397, SEQ ID NO: 408, or SEQ ID NO:419) of the immunologically active fragment. In certain embodiments, anyaddition to the amino terminal of the core of the immunologically activefragment can include one or more amino acid additions, deletions,substitutions (collectively, “modifications”), or any combination ofmodification compared to longer version—e.g., wild-type or other nativeform—of the polypeptide. Thus, for example, in embodiments in which theimmunologically active fragment includes SEQ ID NO:397, an addition tothe amino terminal of SEQ ID NO:397 can include at least 1, at least 2,at least 3, at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, at least25, or at least 26 modifications in the amino terminal addition comparedto, for example, amino acids 1-26 of SEQ ID NO:399. As another example,in embodiments in which the immunologically active fragment includes SEQID NO:408, an addition to the amino terminal of SEQ ID NO:408 caninclude at least 1, at least 2, at least 3, at least 4, or at least 5modifications in the amino terminal addition compared to, for example,amino acids 1-26 of SEQ ID NO:415.

When comparing the amino acid sequence similarity and/or amino acidsequence identity of a reference polypeptide and a candidate polypeptideof a different length (e.g., an immunologically active fragment of thereference polypeptide) the similarity and/or identity may be computedover the full length of the longer polypeptide, counting each amino acidresidue of greater length in the longer polypeptide contributing as amismatch.

In some embodiments, therefore, an isolated polypeptide of the inventioncan include a polypeptide having at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence similarity and/or identity to the amino acid sequence ofSEQ ID NO:397, with the proviso that if the isolated polypeptideincludes one or more additional amino acids at the amino terminal, theone or more additional amino acids include at least one amino aciddeletion or at least one amino acid substitution compared to amino acids1-26 of SEQ ID NO:399.

In other embodiments, an isolated polypeptide of the invention caninclude a polypeptide having at least 98% or at least 99% sequencesimilarity and/or identity to the amino acid sequence of SEQ ID NO:408,with the proviso that if the isolated polypeptide includes one or moreadditional amino acids at the amino terminal, the one or more additionalamino acids include at least one amino acid deletion or at least oneamino acid substitution compared to amino acids 1-5 of SEQ ID NO:415.

As noted above in the section headed Polypeptide sequence similarity andpolypeptide sequence identity, a polypeptide identified by reference tothe amino acid sequence of a particular SEQ ID NO can include apolypeptide with at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% amino acid sequence similarityto the reference amino acid sequence (e.g., the amino acid sequenceprovided in a specified SEQ ID NO:) and/or a polypeptide with at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% amino acid sequence identity to the reference aminoacid sequence (e.g., the amino acid sequence provided in a specified SEQID NO:).

A recombinantly-produced polypeptide may be expressed from a vector thatpermits expression of the polypeptide when the vector is introduced intoan appropriate host cell. A host cell may be constructed to produce oneor more recombinantly-produced polypeptides of the invention and,therefore, can include one more vectors that include at least onepolynucleotide that encodes a polypeptide of the invention. Thus, eachvector can include one or more polynucleotides of the invention—i.e., apolynucleotide that encodes a polypeptide of the invention.

Certain compositions such as, for example, those includingrecombinantly-produced polypeptides, can include a maximum number ofpolypeptides. In some embodiments, the maximum number of polypeptidescan refer to the maximum total number of polypeptides. Certaincompositions can include, for example, no more than 50 polypeptides suchas, for example, no more than 40 polypeptides, no more than 30polypeptides, no more than 25 polypeptides, no more than 20polypeptides, no more than 15 polypeptides, no more than 10polypeptides, no more than eight polypeptides, no more than sevenpolypeptides, no more than six polypeptides, no more than fivepolypeptides, no more than four polypeptides, no more than threepolypeptides, no more than two polypeptides, or no more than onepolypeptide. In other embodiments, a maximum number ofrecombinantly-produced polypeptides may be specified in a similarmanner. In still other embodiments, a maximum number ofnonrecombinantly-produced polypeptides may be specified in a similarmanner.

A composition can include polypeptides isolatable from one microbe, orcan be isolatable from a combination of two or more microbes. Forinstance, a composition can include polypeptides isolatable from two ormore Staphyloccocus spp., or from a Staphyloccocus spp. and a differentmicrobe that is not a member of the genus Staphyloccocus. The presentinvention also provides compositions including a whole cell preparation,where the whole cell expresses one or more of the polypeptides of thepresent invention. For instance, the whole cell can be a Staphyloccocusspp. In some aspects, a composition can include whole preparations fromtwo, three, four, five, or six strains.

Optionally, a polypeptide of the present invention can be covalentlybound or conjugated to a carrier polypeptide to improve theimmunological properties of the polypeptide. Useful carrier polypeptidesare known in the art. The chemical coupling of polypeptides of thepresent invention can be carried out using known and routine methods.For instance, various homobifunctional and/or heterobifunctionalcross-linker reagents such as bis(sulfosuccinimidyl) suberate,bis(diazobenzidine), dimethyl adipimidate, dimethyl pimelimidate,dimethyl superimidate, disuccinimidyl suberate, glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide,sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide, sulfosuccinimidyl4-(N-maleimidomethyl) cycloheane-1-carboxylate, sulfosuccinimidyl4-(p-maleimido-phenyl) butyrate and (1-ethyl-3-(dimethyl-aminopropyl)carbodiimide can be used (see, for instance, Harlow and Lane,Antibodies, A Laboratory Manual, generally and Chapter 5, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., N.Y. (1988)).

The compositions of the present invention optionally further include apharmaceutically acceptable carrier. “Pharmaceutically acceptable”refers to a diluent, carrier, excipient, salt, etc, that is compatiblewith the other ingredients of the composition, and not deleterious tothe recipient thereof. Typically, the composition includes apharmaceutically acceptable carrier when the composition is used asdescribed herein. The compositions of the present invention may beformulated in pharmaceutical preparations in a variety of forms adaptedto the chosen route of administration, including routes suitable forstimulating an immune response to an antigen. Thus, a composition of thepresent invention can be administered via known routes including, forexample, oral; parenteral including intradermal, transcutaneous andsubcutaneous; intramuscular, intravenous, intraperitoneal, etc. andtopically, such as, intranasal, intrapulmonary, intramammary,intravaginal, intrauterine, intradermal, transcutaneous and rectally,etc. It is foreseen that a composition can be administered to a mucosalsurface, such as by administration to the nasal or respiratory mucosa(e.g., via a spray or aerosol), in order to stimulate mucosal immunity,such as production of secretory IgA antibodies, throughout the animal'sbody.

A composition of the present invention can also be administered via asustained or delayed release implant. Implants suitable for useaccording to the invention are known and include, for example, thosedisclosed in Emery and Straub (WO 01/37810 (2001)), and Emery et al.,(WO 96/01620 (1996)). Implants can be produced at sizes small enough tobe administered by aerosol or spray. Implants also can includenanospheres and microspheres.

A composition of the present invention may be administered in an amountsufficient to treat certain conditions as described herein. The amountof polypeptides or whole cells present in a composition of the presentinvention can vary. For instance, the dosage of polypeptides can bebetween 0.01 micrograms (μg) and 300 mg, typically between 0.1 mg and 10mg. When the composition is a whole cell preparation, the cells can bepresent at a concentration of, for instance, 10² bacteria/ml, 10³bacteria/ml, 10⁴ bacteria/ml, 10⁵ bacteria/ml, 10⁶ bacteria/ml, 10⁷bacteria/ml, 10⁸ bacteria/ml, or 10⁹ bacteria/ml. For an injectablecomposition (e.g. subcutaneous, intramuscular, etc.) the polypeptidesmay be present in the composition in an amount such that the totalvolume of the composition administered is 0.5 ml to 5.0 ml, typically1.0 to 2.0 ml. When the composition is a whole cell preparation, thecells are preferably present in the composition in an amount that thetotal volume of the composition administered is 0.5 ml to 5.0 ml,typically 1.0 to 2.0 ml. The amount administered will vary depending onvarious factors including, but not limited to, the specific polypeptideschosen, the weight, physical condition and age of the animal, and theroute of administration. Thus, the absolute weight of the polypeptideincluded in a given unit dosage form can vary widely, and depends uponfactors such as the species, age, weight and physical condition of theanimal, as well as the method of administration. Such factors can bedetermined by one of skill in the art. Other examples of dosagessuitable for the invention are disclosed in Emery et al., (U.S. Pat. No.6,027,736).

The formulations may be conveniently presented in unit dosage form andmay be prepared by methods well known in the art of pharmacy. Methods ofpreparing a composition with a pharmaceutically acceptable carrierinclude the step of bringing the active compound (e.g., a polypeptide orwhole cell of the present invention) into association with a carrierthat constitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing theactive compound into association with a liquid carrier, a finely dividedsolid carrier, or both, and then, if necessary, shaping the product intothe desired formulations.

A composition including a pharmaceutically acceptable carrier can alsoinclude an adjuvant. An “adjuvant” refers to an agent that can act in anonspecific manner to enhance an immune response to a particularantigen, thus potentially reducing the quantity of antigen necessary inany given immunizing composition, and/or the frequency of injectionnecessary in order to generate an adequate immune response to theantigen of interest. Adjuvants may include, for example, IL-1, IL-2,emulsifiers, muramyl dipeptides, dimethyl dioctadecyl ammonium bromide(DDA), avridine, aluminum hydroxide, oils, saponins, alpha-tocopherol,polysaccharides, emulsified paraffins (including, for instance, thoseavailable from under the tradename EMULSIGEN from MVP Laboratories,Ralston, Nebr.), ISA-70, RIM and other substances known in the art. Itis expected that polypeptides of the present invention will haveimmunoregulatory activity and that such polypeptides may be used asadjuvants that directly act as T and/or B cell activators or act onspecific cell types that enhance the synthesis of various cytokines oractivate intracellular signaling pathways. Such polypeptides areexpected to augment the immune response to increase the protective indexof the existing composition.

In another embodiment, a composition of the invention including apharmaceutically acceptable carrier can include a biological responsemodifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-alpha,IFN-gamma, and other cytokines that effect immune cells. An immunizingcomposition can also include other components known in the art such asan antibiotic, a preservative, an anti-oxidant, or a chelating agent.

Methods of Making

The present invention also provides methods for obtaining thepolypeptides described herein. The polypeptides and whole cells of thepresent invention may be isolatable from a member of the familyMicrococcaceae, preferably, Staphylococcus spp., more preferably,Staphylococcus aureus. Other gram positive microbes from whichpolypeptides can be isolated include Corynebacterium spp.,Erysipelothrix spp., Mycobacterium spp., and Erysipelothrix spp.Microbes useful for obtaining polypeptides of the present invention andmaking whole cell preparations are commercially available from adepository such as American Type Culture Collection (ATCC). In addition,such microbes are readily obtainable by techniques routine and known tothe art. The microbes may be derived from an infected animal as a fieldisolate, and used to obtain polypeptides and/or whole cell preparationsof the present invention, or stored for future use, for example, in afrozen repository at −20° C. to −95° C., or −40° C. to −50° C., inbacteriological media containing 20% glycerol, and other like media.

When a polypeptide of the present invention is to be obtained from amicrobe, the microbe can be incubated under low metal conditions. Asused herein, the phrase “low metal conditions” refers to an environment,typically bacteriological media, which contains amounts of a free metalthat cause a microbe to express metal-regulated polypeptides at adetectable level. As used herein, the phrase “high metal conditions”refers to an environment that contains amounts of a free metal thatcause a microbe to either not express one or more of the metal-regulatedpolypeptides described herein at a detectable level, or to express sucha polypeptide at a decreased level compared to expression of themetal-regulated polypeptide under low metal conditions. In some cases,“high metal conditions” can include a metal-rich natural environmentand/or culture in a metal-rich medium without a metal chelator. Incontrast, in some cases, “low metal conditions” can include culture in amedium that includes a metal chelator, as described in more detailbelow. Metals are those present in the periodic table under Groups 1through 17 (IUPAC notation; also referred to as Groups I-A, II-A, IV-B,V-B, VI-B, VII-B, VIII, I-B, II-B, III-A, IV-A, V-A, VI-A, and VII-A,respectively, under CAS notation). Preferably, metals are those inGroups 2 through 12, more preferably, Groups 3-12. Even more preferably,the metal is iron, zinc, copper, magnesium, nickel, cobalt, manganese,molybdenum, or selenium, most preferably, iron.

Low metal conditions are generally the result of the addition of a metalchelating compound to a bacteriological medium, the use of abacteriological medium that contains low amounts of a metal, or thecombination thereof. High metal conditions are generally present when achelator is not present in the medium, a metal is added to the medium,or the combination thereof. Examples of metal chelators include naturaland synthetic compounds. Examples of natural compounds include plantphenolic compounds, such as flavenoids. Examples of flavenoids includethe copper chelators catechin and naringenin, and the iron chelatorsmyricetin and quercetin. Examples of synthetic copper chelators include,for instance, tetrathiomolybdate, and examples of synthetic zincchelators include, for instance, N,N,N′,N′-Tetrakis(2-pyridylmethyl)-ethylene diamine. Examples of synthetic iron chelatorsinclude 2,2′-dipyridyl (also referred to in the art as α,α′-bipyridyl),8-hydroxyquinoline, ethylenediamine-di-O-hydroxyphenylacetic acid(EDDHA), desferrioxamine methanesulphonate (desferol), transferrin,lactoferrin, ovotransferrin, biological siderophores, such as, thecatecholates and hydroxamates, and citrate. An example of a generaldivalent cation chelator is CHELEX resin. Preferably, 2,2′-dipyridyl isused for the chelation of iron. Typically, 2,2′-dipyridyl is added tothe media at a concentration of at least 300 micrograms/milliliter(μg/ml), at least 600 μg/ml, or at least 900 μg/ml. High levels of2,2′-dipyridyl can be 1200 μg/ml, 1500 μg/ml, or 1800 μg/ml.

The S. aureus genome encodes three Fur homologs: Fur, PerR, and Zur.While the Zur and PerR proteins appear to be primarily involved inregulating zinc homeostasis and peroxide stress genes, respectively, theFur protein has been demonstrated to regulate several iron-siderophoreuptake systems in response to iron limitation. The Fur protein alsoplays a role in oxidative stress resistance and virulence. It isexpected that a gram positive organism, preferably, an S. aureus, with amutation in a fur gene will result in the constitutive expression ofmany, if not all, of the metal-regulated polypeptides of the presentinvention. The production of a fur mutation in a gram positive,preferably, an S. aureus, can be produced using routine methodsincluding, for instance, transposon, chemical, or site-directedmutagenesis useful for generating gene knock-out mutations in grampositive bacteria.

The medium used to incubate the microbe and the volume of media used toincubate the microbe can vary. When a microbe is being evaluated for theability to produce one or more of the polypeptides described herein, themicrobe can be grown in a suitable volume, for instance, 10 millilitersto 1 liter of medium. When a microbe is being grown to obtainpolypeptides for use in, for instance, administration to animals, themicrobe may be grown in a fermentor to allow the isolation of largeramounts of polypeptides. Methods for growing microbes in a fermentor areroutine and known to the art. The conditions used for growing a microbepreferably include a metal chelator, more preferably an iron chelator,for instance 2,2′-dipyridyl, a pH of between 6.5 and 7.5, preferablybetween 6.9 and 7.1, and a temperature of 37° C.

In some aspects of the invention, a microbe may be harvested aftergrowth. Harvesting includes concentrating the microbe into a smallervolume and suspending in a media different than the growth media.Methods for concentrating a microbe are routine and known in the art,and include, for example, filtration or centrifugation. Typically, theconcentrated microbe is suspended in an appropriate buffer. An exampleof a buffer that can be used contains Tris-base (7.3 grams/liter), at apH of 8.5. Optionally, the final buffer also minimizes proteolyticdegradation. This can be accomplished by having the final buffer at a pHof greater than 8.0, preferably, at least 8.5, and/or including one ormore proteinase inhibitors (e.g., phenylmethanesulfonyl fluoride).Optionally and preferably, the concentrated microbe is frozen at −20° C.or below until disrupted.

When the microbe is to be used as a whole cell preparation, theharvested cells may be processed using routine and known methods toinactivate the cells. Alternatively, when a microbe is to be used toprepare polypeptides of the present invention, the microbe may bedisrupted using chemical, physical, or mechanical methods routine andknown to the art, including, for example, boiling, french press,sonication, digestion of peptidoglycan (for instance, by digestion withlysozyme), or homogenization. An example of a suitable device useful forhomogenization is a model C500-B AVESTIN homogenizer, (Avestin Inc,Ottawa Canada). As used herein, “disruption” refers to the breaking upof the cell. Disruption of a microbe can be measured by methods that areroutine and known to the art, including, for instance, changes inoptical density. Typically, a microbe is subjected to disruption untilthe percent transmittance is increased by 20% when a 1:100 dilution ismeasured. When physical or mechanical methods are used, the temperatureduring disruption is typically kept low, preferably at 4° C., to furtherminimize proteolytic degradation. When chemical methods are used thetemperature may be increased to optimize for the cell disruption. Acombination of chemical, physical, and mechanical methods may also beused to solubilize the cell wall of microbe. As used herein, the term“solubilize” refers to dissolving cellular materials (e.g.,polypeptides, nucleic acids, carbohydrates) into the aqueous phase ofthe buffer in which the microbe was disrupted, and the formation ofaggregates of insoluble cellular materials. Without intending to belimited by theory, the conditions for solubilization are believed toresult in the aggregation of polypeptides of the present invention intoinsoluble aggregates that are large enough to allow easy isolation by,for instance, centrifugation.

The insoluble aggregates that include one or more of the polypeptides ofthe present invention may be isolated by methods that are routine andknown to the art. Preferably, the insoluble aggregates are isolated bycentrifugation. Typically, centrifugation of polypeptides, such asmembrane polypeptides, can be accomplished by centrifugal forces of100,000×g. The use of such centrifugal forces requires the use ofultracentrifuges, and scale-up to process large volumes of sample isoften difficult and not economical with these types of centrifuges. Themethods described herein provide for the production of insolubleaggregates large enough to allow the use of continuous flow centrifuges,for instance T-1 Sharples (Alfa Laval Separations, Warminster, Pa.),which can be used with a flow rate of 250 ml/minute at 17 psi at acentrifugal force of 46,000×g to 60,000×g. Other large scale centrifugescan be used, such as the tubular bowl, chamber, and disc configurations.Such centrifuges are routinely used and known in the art, and arecommercially available from such manufactures as Pennwalt, Westfalia andalpha-Laval.

The final harvested proteins are washed and/or dialyzed against anappropriate buffer using methods known in the art, for instancediafiltration, precipitation, hydrophobic chromatography, ion-exchangechromatography, or affinity chromatography, or ultra filtration andwashing the polypeptides, for instance, in alcohol, by diafiltration.After isolation, the polypeptides suspended in buffer and stored at lowtemperature, for instance, −20° C. or below.

In those aspects of the present invention where a whole cell preparationis to be made, after growth a microbe can be killed with the addition ofan agent such as glutaraldehyde, formalin, or formaldehyde, at aconcentration sufficient to inactivate the cells in the culture. Forinstance, formalin can be added at a concentration of 0.3% (vol:vol).After a period of time sufficient to inactivate the cells, the cells canbe harvested by, for instance, diafiltration and/or centrifugation, andwashed.

In other aspects, an isolated polypeptide of the invention may beprepared recombinantly. When prepared recombinantly, a polynucleotideencoding the polypeptide may be identified and cloned into anappropriate expression host as described below in Example 14. Therecombinant expression host may be grown in an appropriate medium,disrupted, and the polypeptides isolated as described above.

Methods of Use

An aspect of the present invention is further directed to methods ofusing the compositions of the present invention. The methods includeadministering to an animal an effective amount of a composition of thepresent invention. The animal can be, for instance, avian (including,for instance, chickens or turkeys), bovine (including, for instance,cattle), caprine (including, for instance, goats), ovine (including, forinstance, sheep), porcine (including, for instance, swine), bison(including, for instance, buffalo), equine (including, for instance,horses), a companion animal (including, for instance, dogs or cats),members of the family Cervidae (including, for instance, deer, elk,moose, caribou and reindeer), or human.

In some aspects, the methods may further include additionaladministrations (e.g., one or more booster administrations) of thecomposition to the animal to enhance or stimulate a secondary immuneresponse. A booster can be administered at a time after the firstadministration, for instance, one to eight weeks, preferably two to fourweeks, after the first administration of the composition. Subsequentboosters can be administered one, two, three, four, or more timesannually. Without intending to be limited by theory, it is expected thatin some aspects of the present invention annual boosters will not benecessary, as an animal will be challenged in the field by exposure tomicrobes expressing polypeptides present in the compositions havingepitopes that are identical to or structurally related to epitopespresent on polypeptides of the composition administered to the animal.

In one aspect, the invention is directed to methods for makingantibodies, for instance by inducing the production of antibody in ananimal, or by recombinant techniques. The antibody produced includesantibody that specifically binds at least one polypeptide present in thecomposition. In this aspect of the invention, an “effective amount” isan amount effective to result in the production of antibody in theanimal. Methods for determining whether an animal has producedantibodies that specifically bind polypeptides present in a compositionof the present invention can be determined as described herein. Thepresent invention further includes antibody that specifically bind to apolypeptide of the present invention, and compositions including suchantibodies.

The method may be used to produce antibody that specifically bindspolypeptides expressed by a microbe other than the microbe from whichthe polypeptides of the composition were isolated. As used herein, anantibody that can “specifically bind” a polypeptide is an antibody thatinteracts with the epitope of the antigen that induced the synthesis ofthe antibody, or interacts with a structurally related epitope. At leastsome of the polypeptides present in the compositions of the presentinvention typically include epitopes that are conserved in thepolypeptides of different species and different genera of microbes.Accordingly, antibody produced using a composition derived from onemicrobe is expected to bind to polypeptides expressed by other microbesand provide broad spectrum protection against gram positive organisms.Examples of gram positive microbes to which the antibody mayspecifically bind are Micrococcaceae, preferably, Staphylococcus spp.,more preferably, Staphylococcus aureus; members of the familyStreptococcaceae, preferably, Streptococcus pyogenes, Streptococcuspneumoniae, Streptococcus agalactiae, Streptococcus uberis,Streptococcus bovis, Streptococcus equi, or Streptococcus dysgalactiae;and Bacillus spp., Clostridium spp., Corynebacterium spp., Enterococcusspp., Erysipelothrix spp., Listeria spp., Micrococcus spp., andMycobacterium spp., Kytococcus spp., and Erysipelothrix spp. Therefore,antibody produced using a composition of polypeptides of the inventionmay be used to identify and characterize polypeptides of the inventionindependent of the origin, source, and/or manner of obtaining thepolypeptide.

The present invention is also directed to the use of such antibody totarget a microbe expressing a polypeptide of the present invention or apolypeptide having an epitope structurally related to an epitope presenton a polypeptide of the present invention. A compound can be covalentlybound to an antibody, where the compound can be, for instance, a toxin.Likewise, such compounds can be covalently bound to a bacterialsiderophore to target the microbe. The chemical coupling or conjugationof an antibody of the present invention, or a portion thereof (such asan Fab fragment), can be carried out using known and routine methods.

In one aspect the invention is also directed to treating an infection inan animal, including a human, caused by a gram positive microbe,preferably by a member of the family Micrococcaceae, preferably,Staphylococcus spp., more preferably, S. aureus; members of the familyStreptococcaceae, preferably, Streptococcus pyogenes, Streptococcuspneumoniae, Streptococcus agalactiae, Streptococcus uberis,Streptococcus bovis, Streptococcus equi, or Streptococcus dysgalactiae;Bacillus spp., Clostridium spp., Corynebacterium spp., Enterococcusspp., Erysipelothrix spp., Kytococcus spp., Listeria spp., Micrococcusspp., Mycobacterium spp., and Erysipelothrix spp. As used herein, theterm “infection” refers to the presence of a gram positive microbe in ananimal's body, which may or may not be clinically apparent. An animalwith an infection by a member of the genus Staphylococcus that is notclinically apparent is often referred to as an asymptomatic carrier.

Treating an infection can be prophylactic or, alternatively, can beinitiated after the animal is infected by the microbe. Treatment that isprophylactic—e.g., initiated before a subject is infected by a microbeor while any infection remains subclinical—is referred to herein astreatment of a subject that is “at risk” of infection. As used herein,the term “at risk” refers to an animal that may or may not actuallypossess the described risk. Thus, typically, an animal “at risk” ofinfection by a microbe is an animal present in an area where animalshave been identified as infected by the microbe and/or is likely to beexposed to the microbe even if the animal has not yet manifested anydetectable indication of infection by the microbe and regardless ofwhether the animal may harbor a subclinical amount of the microbe.Accordingly, administration of a composition can be performed before,during, or after the animal has first contact with the microbe.Treatment initiated after the animal's first contact with the microbemay result in decreasing the severity of symptoms and/or clinical signsof infection by the microbe, completely removing the microbe, and/ordecreasing the likelihood of experiencing a clinically evident infectioncompared to an animal to which the composition is not administered. Themethod includes administering an effective amount of the composition ofthe present invention to an animal having, or at risk of having, aninfection caused by a gram positive microbe, and determining whether thenumber of microbes causing the infection has decreased. In this aspectof the invention, an “effective amount” is an amount effective to reducethe number of the specified microbes in an animal or reduce thelikelihood that the animal experiences a clinically-evident infectioncompared to an animal to which the composition is not administered.Methods for determining whether an infection is caused by a grampositive microbe are routine and known in the art, as are methods fordetermining whether the infection has decreased.

In another aspect, the present invention is directed to methods fortreating one or more symptoms or clinical signs of certain conditions inan animal that may be caused by infection by a gram positive microbe,preferably by a member of the family Micrococcaceae, preferably,Staphylococcus spp., more preferably, S. aureus; members of the familyStreptoococcaceae, preferably, Streptococcus pyogenes, Streptococcuspneumoniae, Streptococcus agalactiae, Streptococcus uberis,Streptococcus bovis, Streptococcus equi, or Streptococcus dysgalactiae;Bacillus spp., Clostridium spp., Corynebacterium spp., Enterococcusspp., Erysipelothrix spp., Kytococcus spp., Listeria spp., Micrococcusspp., Mycobacterium spp., and Erysipelothrix spp. The method includesadministering an effective amount of a composition of the presentinvention to an animal having or at risk of having a condition, orexhibiting symptoms and/or clinical signs of a condition, anddetermining whether at least one symptom and/or clinical sign of thecondition is changed, preferably, reduced. Examples of conditions and/orclinical signs caused by microbial infections include, for instance,mastitis, septicemia, pneumonia, meningoencephalitis, lymphangitis,dermatitis, genital tract infections, strangles, metritis, perinataldisease, pituitary abscesses, arthritis, bursitis, orchitis, cystitisand pyelonephritis, caseous lymphadenitis, tuberculosis, ulcerativelymphangitis, listeriosis, erysipelas, laminitis, anthrax, tyzzer'sdisease, tetanus, botulism, enteritis, malignant edema, braxy, bacillaryhemoglobinuria, enterotoxemia, necrotic skin lesions, and nosocomialinfections. Examples of conditions caused by S. aureus also include, forinstance, botryomycosis in horses, purulent synovitis and osteomyelitisin poultry, abortions in swine, and tick pyemia in lambs. Examples ofconditions caused by Streptococcus spp. also include, for instance, sorethroat, scarlet fever, impetigo, ulcerative endocarditis, rheumaticfever and post streptococcal glomerulonephritis cervicitis in humans,cervicitis in equine and swine, and meningitis and jowl abscesses inswine.

Treatment of symptoms and/or clinical signs associated with theseconditions can be prophylactic or, alternatively, can be initiated afterthe development of a condition described herein. As used herein, theterm “symptom” refers to subjective evidence of disease or conditionexperienced by the patient and caused by infection by a microbe. As usedherein, the term “clinical sign” or, simply, “sign” refers to objectiveevidence of disease or condition caused by infection by a microbe.Symptoms and/or clinical signs associated with conditions referred toherein and the evaluations of such symptoms are routine and known in theart. Treatment that is prophylactic, for instance, initiated before asubject manifests symptoms or signs of a condition caused by a microbe,is referred to herein as treatment of a subject that is “at risk” ofdeveloping the condition. Thus, typically, an animal “at risk” ofdeveloping a condition is an animal present in an area where animalshaving the condition have been diagnosed and/or is likely to be exposedto a microbe causing the condition even if the animal has not yetmanifested symptoms or signs of any condition caused by the microbe.Accordingly, administration of a composition can be performed before,during, or after the occurrence of the conditions described herein.Treatment initiated after the development of a condition may result indecreasing the severity of the symptoms of one of the conditions, orcompletely removing the symptoms. In this aspect of the invention, an“effective amount” is an amount effective to prevent the manifestationof symptoms of a disease, decrease the severity of the symptoms of adisease, and/or completely remove the symptoms. The successful treatmentof a gram positive microbial infection in an animal is disclosed inExample 5, which demonstrates the protection against disease caused byS. aureus in mouse models by administering a composition of the presentinvention. These mouse models are a commonly accepted model for thestudy of human disease caused by these microbes. The successfultreatment of a gram positive microbial infection in an animal is alsodisclosed in Examples 10 to 12, which demonstrate that administering acomposition of the present invention provides protection against diseasecaused by S. aureus in cows.

The present invention also provides methods for decreasing colonizationby gram positive microbes, for instance blocking the attachment sites ofgram positive microbe, including tissues of the skeletal system (forinstance, bones, cartilage, tendons and ligaments), muscular system,(for instance, skeletal and smooth muscles), circulatory system (forinstance, heart, blood vessels, capillaries and blood), nervous system(for instance, brain, spinal cord, and peripheral nerves), respiratorysystem (for instance, nose, trachea lungs, bronchi, bronchioceles,alveoli), digestive system (for instance, mouth, salivary glandsoesophagus liver stomach large and small intestine), excretory system(for instance, kidneys, ureters, bladder and urethra), endocrine system(for instance, hypothalamus, pituitary, thyroid, pancreas and adrenalglands), reproductive system (for instance, ovaries, oviduct, uterus,vagina, mammary glands, testes, and seminal vesicles), lymphatic/immunesystems (for instance, lymph, lymph nodes and vessels, mononuclear orwhite blood cells, such as macrophages, neutrophils, monocytes,eosinophils, basophils, and lymphocytes, including T cells and B cells),and specific cell lineages (for instance, precursor cells, epithelialcells, stem cells), and the like. Preferably, the gram positive microbeis a member of the family Micrococcaceae, preferably, Staphylococcusspp., more preferably, S. aureus; a member of the familyStreptooccaceae, preferably, Streptococcus pyogenes, Streptococcuspneumoniae, Streptococcus agalactiae, Streptococcus uberis,Streptococcus bovis, Streptococcus equi, or Streptococcus dysgalactiae;Bacillus spp., Clostridium spp., Corynebacterium spp., Enterococus spp.,Erysipelothrix spp., Kytococcus spp., Listeria spp., Micrococcus spp.,Mycobacterium spp., and Erysipelothrix spp.

Decreasing colonization in an animal may be performed prophylacticallyor, alternatively, can be initiated after the animal is colonized by themicrobe. Treatment that is prophylactic—e.g., initiated before a subjectis colonized by a microbe or while any colonization remainsundetected—is referred to herein as treatment of a subject that is “atrisk” of colonization by the microbe. Thus, typically, an animal “atrisk” of colonization by a microbe is an animal present in an area whereanimals have been identified as colonized by the microbe and/or islikely to be exposed to the microbe even if the animal has not yetmanifested any detectable indication of colonization by the microbe andregardless of whether the animal may harbor a subcolonization number ofthe microbe. Accordingly, administration of a composition can beperformed before, during, or after the animal has first contact with themicrobe. Treatment initiated after the animal's first contact with themicrobe may result in decreasing the extent of colonization by themicrobe, completely removing the microbe, and/or decreasing thelikelihood that the animal becomes colonized by the microbe compared toan animal to which the composition is not administered. Thus, the methodincludes administering an effective amount of a composition of thepresent invention to an animal colonized by, or at risk of beingcolonized by, a gram positive microbe. In this aspect of the invention,an “effective amount” is an amount sufficient to decrease colonizationof the animal by the microbe, where decreasing colonization refers toone or more of: decreasing the extent of colonization by the microbe,completely removing the microbe, and/or decreasing the likelihood thatthe animal becomes colonized by the microbe compared to an animal towhich the composition is not administered. Methods for evaluating thecolonization of an animal by a microbe are routine and known in the art.For instance, colonization of an animal's intestinal tract by a microbecan be determined by measuring the presence of the microbe in theanimal's feces. It is expected that decreasing the colonization of ananimal by a microbe will reduce transmission of the microbe to humans.

A composition of the invention can be used to provide for active orpassive immunization against bacterial infection. Generally, thecomposition can be administered to an animal to provide activeimmunization. However, the composition can also be used to induceproduction of immune products, such as antibodies, which can becollected from the producing animal and administered to another animalto provide passive immunity. Immune components, such as antibodies, canbe collected to prepare compositions (preferably containing antibody)from serum, plasma, blood, colostrum, etc. for passive immunizationtherapies. Antibody compositions including monoclonal antibodies and/oranti-idiotypes can also be prepared using known methods. Chimericantibodies include human-derived constant regions of both heavy andlight chains and murine-derived variable regions that areantigen-specific (Morrison et al., Proc. Natl. Acad. Sci. USA, 1984,81(21):6851-5; LoBuglio et al., Proc. Natl. Acad. Sci. USA, 1989,86(11):4220-4; Boulianne et al., Nature, 1984, 312(5995):643-6.).Humanized antibodies substitute the murine constant and framework (FR)(of the variable region) with the human counterparts (Jones et al.,Nature, 1986, 321(6069):522-5; Riechmann et al., Nature, 1988,332(6162):323-7; Verhoeyen et al., Science, 1988, 239(4847):1534-6;Queen et al., Proc. Natl. Acad. Sci. USA, 1989, 86(24):10029-33;Daugherty et al., Nucleic Acids Res., 1991, 19(9): 2471-6.).Alternatively, certain mouse strains can be used that have beengenetically engineered to produce antibodies that are almost completelyof human origin; following immunization the B cells of these mice areharvested and immortalized for the production of human monoclonalantibodies (Bruggeman and Taussig, Curr. Opin. Biotechnol., 1997,8(4):455-8; Lonberg and Huszar, Int. Rev. Immunol., 1995; 13(1):65-93;Lonberg et al., Nature, 1994, 368:856-9; Taylor et al., Nucleic AcidsRes., 1992, 20:6287-95.). Passive antibody compositions and fragmentsthereof, e.g., scFv, Fab, F(ab′)₂ or Fv or other modified forms thereof,may be administered to a recipient in the form of serum, plasma, blood,colostrum, and the like. However, the antibodies may also be isolatedfrom serum, plasma, blood, colostrum, and the like, using known methodsfor later use in a concentrated or reconstituted form such as, forinstance, lavage solutions, impregnated dressings and/or topical agentsand the like. Passive immunization preparations may be particularlyadvantageous for the treatment of acute systemic illness, or passiveimmunization of young animals that failed to receive adequate levels ofpassive immunity through maternal colostrum. Antibodies useful forpassive immunization may also be useful to conjugate to various drugs orantibiotics that could be directly targeted to bacteria expressingduring a systemic or localized infection a polypeptide of the presentinvention or a polypeptide having an epitope structurally related to anepitope present on a polypeptide of the present invention.

Animal models, in particular mouse models, are available forexperimentally evaluating the compositions of the present invention.These mouse models are commonly accepted models for the study of humandisease caused by members of the genus Staphylococcus, and S. aureus inparticular. In those cases where a member of the genus Staphylococcuscauses disease in an animal, for instance a cow, the natural host can beused to experimentally evaluate the compositions of the presentinvention.

However, protection in a mouse model is not the only way to assesswhether a composition can confer protection to an animal againstinfection by a Staphylococcus spp. The adaptive immune response consistsof two primary divisions: the humoral (antibody) response and thecellular (T cell) response. Following infection by a bacterial pathogen,dendritic cells at the infection site encounter microbial antigens andproduce signaling molecules such as, for example, surface receptors andcytokines in response to conserved molecular patterns associated withthe specific bacterium. These signals are shaped by the nature of thepathogen and ideally lead to the appropriate antibody and T cellresponses that protect the host from disease. While some bacterialdiseases are controlled primarily through antibody functions, othersrequire T cell responses or both antibody and T cell responses forprotection. The goal of vaccine biology is to identify the immuneresponses that provide protection and then design a vaccine to reproduceone or more of these responses in humans.

Antibodies can have many different functions in the conferringprotection against infection such as, for example, complement fixation,opsonization, neutralization, and/or agglutination. Moreover, somesubclasses of antibodies are better than others at specific functions;for example, for complement fixation the following hierarchy exists forhuman IgG subclasses: IgG3>IgG1>IgG2>IgG4).

Antibody immunological functions can be studied in a variety of ways.For instance, Western blots are used to identify antigen-specificbinding based on size of separated proteins, while the standardenzyme-linked immunosorbant assay (ELISA) is used to producequantitative information about antibody titers within serum. Antibodysurface binding studies are used to determine whether antibody in serumare able to recognize antigens on the surface of intact bacteria, animportant indicator of whether the antibodies have the potential to workin vivo. Thus, one skilled in the art recognizes that antibody bindingassays such as a Western blot, ELISA (e.g., using human antisera),and/or surface binding correlate positively with the specifically-boundantigens providing immunological activity against infection byStaphylococcus spp. (Vytvytska et al. 2002, Proteomics 2:580-590; Kuklinet al. 2006, Infect. Immun. 74(4):2215-2223; Dryla et al. 2005, Clin.Diag. Lab. Immunol. 12(3):387-398). However, one skilled in the artfurther recognizes that a lack of antibody binding in an assay such as,for example, a Western blot, ELISA, or surface binding assay does notmean that the assayed antigen fails to provide immunological activityagainst infection by Staphylococcus spp. (Kim H K et al. IsdA and IsdBantibodies protect mice against Staphylococcus aureus abcess formationand lethal challenge. Vaccine (2010),doi:10.1016/j.vaccine.2010.02.097).

FIG. 202 shows that convalescent mouse serum binds to at leastrecombinantly-produced MntC (SEQ ID NO:419), recombinantly-produced SYN2(SEQ ID NO:386), recombinantly-produced SirA (SEQ ID NO:375), andrecombinantly-produced Opp1A (SEQ ID NO:364), indicating that each ofthe bound recombinantly-produced polypeptides can induce immunologicalactivity against infection by Staphylococcus spp.

FIG. 204 shows that convalescent human serum binds torecombinantly-produced PflB (SEQ ID NO:353), recombinantly-producedOpp1A (SEQ ID NO:364), recombinantly-produced SirA (SEQ ID NO:375),recombinantly-produced SYN2 (SEQ ID NO:386), recombinantly-produced FhuD(SEQ ID NO:397), recombinantly-produced SYN1 (SEQ ID NO:408), andrecombinantly-produced MntC (SEQ ID NO:419), indicating that each of therecombinantly-produced polypeptides can induce immunological activityagainst infection by Staphylococcus spp.

FIG. 205 shows that antibody raised against recombinantly-produced FhuD(SEQ ID NO:397), recombinantly-produced Opp1A (SEQ ID NO:364), andrecombinantly-produced PflB (SEQ ID NO:353) binds to the surface ofStaphylococcus spp. cells, indicating that each of the polypeptidetargets of the cell-binding antibody can induce immunological activityagainst infection by Staphylococcus spp.

Techniques such as opsonophagocytosis assays (OPA), in which antibodyand complement-bound bacteria are combined with human or mousephagocytes to determine levels of bacterial killing, are useful forstudying antibody function. Positive OPA results correlate withvaccine-induced protection in a mouse model. (Stranger-Jones et al.2006, Proc. Natl. Acad. Sci. 103(45):16942-16947). A similar oxidativeburst assay can be used to assess the level of reactive oxygen species(ROS) by fresh human or mouse neutrophils following interaction withantibody and complement-bound bacteria.

In some cases, one can determine that a candidate polypeptide possessescell-mediated immunological activity and, therefore, the candidatepolypeptide may exhibit immunological activity in the absence ofinducing the production of antibodies. (Spellberg et al. 2008, Infect.Immun. 76(10):4575-4580). Cytotoxic or CD8 T cells primarily killinfected cells directly through various effector mechanisms, whilehelper CD4 T cells function to provide important signaling in the way ofcytokines. These T cell classes can be further subdivided based on thecytokines they produce, and different subclasses are effective againstdifferent bacterial pathogens. T cells are often studied by assessingtheir phenotypes with flow cytometry, where antibodies are used tovisualize the levels of specific surface markers that enableclassification of the T cells as, for example, a recently activated CD4⁺T cell, a memory CD8⁺ T cell, etc. In addition, cytokines and otherproducts of T cells can be studied by isolating the T cells fromlymphoid tissue and restimulating them with cognate antigen. Followingantigen stimulation the T cells produce cytokines that may be visualizedby, for example, intracellular cytokine staining coupled with flowcytometry, or collecting the cell supernatants and using Luminex beadtechnology to measure 15-25 cytokines simultaneously.

FIG. 206 shows that a composition (rSIRP7) that includesrecombinantly-produced PflB (SEQ ID NO:353), recombinantly-producedOpp1A (SEQ ID NO:364), recombinantly-produced SirA (SEQ ID NO:375),recombinantly-produced SYN2 (SEQ ID NO:386), recombinantly-produced FhuD(SEQ ID NO:397), recombinantly-produced SYN1 (SEQ ID NO:408), andrecombinantly-produced MntC (SEQ ID NO:419) induces a cytokine profilesimilar to the cytokine profile induced by the SIRP extract demonstratedto provide immunological activity against infection by Staphylococcusspp. The rSIRP7 composition induced the production of, for example,IL-2, IL-6, IL-17, IFN-γ, MIP-2, and GM-CSF.

Thus, in addition to mouse models, those of ordinary skill in the artrecognize that immunological activity commensurate with the methodsdescribed herein may correlate with any one or more of the following:Western blot data showing that serum from animals exposed to aSyaphylococcus spp. contains antibody that specifically binds to acandidate polypeptide, cell surface binding assays demonstrating thatantibody that specifically binds to a candidate polypeptide specificallybinds to a Staphylococcus spp., opsonophagocytosis data, and cytokineinduction.

Another aspect of the present invention provides methods for detectingantibody that specifically binds polypeptides of the present invention.These methods are useful in, for instance, detecting whether an animalhas antibody that specifically binds polypeptides of the presentinvention, and diagnosing whether an animal may have a condition causedby a microbe expressing polypeptides described herein, or expressingpolypeptides that share epitopes with the polypeptides described herein.Such diagnostic systems may be in kit form. The methods includecontacting an antibody with a preparation that includes a polypeptide ofthe present invention to result in a mixture. The antibody may bepresent in a biological sample, for instance, blood, milk, or colostrum.The method further includes incubating the mixture under conditions toallow the antibody to specifically bind the polypeptide to form apolypeptide:antibody complex. As used herein, the term“polypeptide:antibody complex” refers to the complex that results whenan antibody specifically binds to a polypeptide. The preparation thatincludes the polypeptides of the present invention may also includereagents, for instance a buffer, that provide conditions appropriate forthe formation of the polypeptide:antibody complex. Thepolypeptide:antibody complex is then detected. The detection ofantibodies is known in the art and can include, for instance,immunofluorescence or peroxidase. The methods for detecting the presenceof antibodies that specifically bind to polypeptides of the presentinvention can be used in various formats that have been used to detectantibody, including radioimmunoassay and enzyme-linked immunosorbentassay.

The present invention also provides a kit for detecting antibody thatspecifically binds polypeptides of the present invention. The antibodydetected may be obtained from an animal suspected to have an infectioncaused by a gram positive microbe, more preferably, a member of thefamily Micrococcaceae, preferably, Staphylococcus spp., more preferably,S. aureus; Streptococcus spp., Bacillus spp., Clostridium spp.,Corynebacterium spp., Enterococus spp., Erysipelothrix spp., Kytococcusspp., Listeria spp., Micrococcus spp., Mycobacterium spp., andErysipelothrix spp.

The kit includes at least one of the polypeptides of the presentinvention (e.g., one, at least two, at least three, etc.), in a suitablepackaging material in an amount sufficient for at least one assay.Optionally, other reagents such as buffers and solutions needed topractice the invention are also included. For instance, a kit may alsoinclude a reagent to permit detection of an antibody that specificallybinds to a polypeptide of the present invention, such as a detectablylabeled secondary antibody designed to specifically bind to an antibodyobtained from an animal. Instructions for use of the packagedpolypeptides are also typically included. As used herein, the phrase“packaging material” refers to one or more physical structures used tohouse the contents of the kit. The packaging material is constructed bywell known methods, generally to provide a sterile, contaminant-freeenvironment. The packaging material may have a label which indicatesthat the polypeptides can be used for detecting antibody thatspecifically binds polypeptides of the present invention. In addition,the packaging material contains instructions indicating how thematerials within the kit are employed to detect the antibody. As usedherein, the term “package” refers to a container such as glass, plastic,paper, foil, and the like, capable of holding within fixed limits thepolypeptides, and other reagents, for instance a secondary antibody.Thus, for example, a package can be a microtiter plate well to whichmicrogram quantities of polypeptides have been affixed. A package canalso contain a secondary antibody. “Instructions for use” typicallyinclude a tangible expression describing the reagent concentration or atleast one assay method parameter, such as the relative amounts ofreagent and sample to be admixed, maintenance time periods forreagent/sample admixtures, temperature, buffer conditions, and the like.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Preparation of Iron Regulated Proteins LaboratoryScale

Compositions derived from different strains of Staphylococcus aureusincluding novel proteins expressed under iron-restriction and/or otherdegrees of metal ion chelation were evaluated for efficacy against avirulent challenge in mice. The efficacy of the composition wasevaluated by collecting data on the following parameters (1) theefficacy of each composition to provide homologous and heterologousprotection against a live virulent challenge in mice, (2) the efficacyof each composition to reduce necrotic skin lesions, and (3) theefficacy of compositions derived from Staphylococcus grown in repleteand deplete iron conditions to provide protection.

The Staphylococcus aureus strains evaluated in this study originatedfrom three animal species; avian, human and bovine. The avian isolateSAAV1 was a field isolate originating from a flock of diseased turkeyshaving a high degree of osteomyelitis and synovitis. The bovine isolates(strain 1477 and strain 2176) were isolated from two differentcommercial dairy herds having a high incidence of clinical mastitis. Thehuman isolate was obtained from the ATCC (strain 19636), and originatedfrom a patient having clinical osteomyelitis.

Master seed stocks of each isolate were prepared by inoculating theappropriate isolate into 200 ml of Tryptic Soy Broth (TSB, DifcoLaboratories, Detroit, Mich.) containing 300 μM 2,2-dipyridyl(Sigma-Aldrich St. Louis, Mo.). The culture was grown while stirring at200 rpm for 6 hours at 37° C., and collected by centrifugation at10,000×g. The bacterial pellet was re-suspended into 100 ml TSB brothcontaining 20% glycerol, and sterilely dispensed into 2 ml cryogenicvials (1 ml per vial) and stored at −90° C. until use.

Each master seed stock was expanded into a working seed. One vial ofeach master seed isolate was inoculated into 200 ml of Tryptic Soy Broth(TSB, Difco Laboratories, Detroit, Mich.) containing 1000 μM2,2-dipyridyl (Sigma-Aldrich St. Louis, Mo.). The culture was grownwhile stirring at 200 rpm for 6 hours at 37° C., and collected bycentrifugation at 10,000×g. The bacterial pellet was resuspended into100 ml TSB broth containing 20% glycerol, and sterilely dispensed into 2ml cryogenic vials (1 ml per vial) and stored at −90° C. until use. Theworking seed was used for the production of compositions enriched withiron-regulated membrane proteins, including iron-regulated membraneproteins.

All strains were adapted to grow in highly iron-depleted media (i.e.,media containing very low levels of free iron). This was accomplished bysub-culturing the bacteria in TSB containing increasing concentrationsof 2,2-dipyridyl (from 300 to 1600 μM).

Proteins were prepared from bacteria as follows. The bacteria were grownfrom frozen working seed stocks by subculturing into 25 ml ofiron-deplete media (containing 1000 μM 2,2′-dyipyridyl) and iron-repletemedia, then incubated at 37° C. while shaking at 400 rpm. Following 12hours of incubation, 5 ml of each culture was transferred into 500 ml ofiron-deplete or iron-replete media pre-incubated at 37° C. Cultures wereincubated for 8 hours at 37° C. while shaking at 100 rpm, then cellswere pelleted by centrifugation at 10,000×g for 20 minutes. Bacterialpellets were resuspended in 100 ml of sterile physiological saline andcentrifuged at 10,000×g for 10 minutes. Pellets were then resuspended in45 ml of Tris-buffered saline, pH 7.2 (TBS; 25 mM Tris, 150 mM NaCl) andthe resulting bacterial suspensions were dispensed as 9-ml aliquots into5 individual tubes. One milliliter of TBS containing 50 units oflysostaphin (Sigma, St. Louis, Mo.) was added to each tube to give afinal volume of 5 units/ml. Following incubation at 37° C. for 30minutes while shaking at 200 rpm, 1 ml of TBS containing 0.1 mg oflysozyme (Sigma) was added to each tube. The bacterial suspensions werethen incubated for an additional 45 minutes while shaking at 200 rpm.Next, suspensions were centrifuged at 3050×g for 12 minutes at 4° C. topellet large cellular debris. The supernatants were collected byaspiration without disturbing the pellet. The supernatant was thencentrifuged at 39,000×g for 2.5 hours. The resulting pellets containingthe proteins were resusupended into 200 μL Tris buffer, pH 7.2, withoutsaline. The protein solution for each isolate were combined for a totalvolume of 1 ml and stored at −90° C.

The protein-enriched extracts derived from S. aureus weresize-fractionated on SDS-PAGE gels using a 4% stacking gel and 10%resolving gel. Samples for electrophoresis were prepared by combining 10μl of sample with 30 μl of SDS reducing sample buffer (62.5 mM Tris-HCLpH 6.8, 20% glycerol, 2% SDS, 5% β-mercaptoethanol) and boiled for 4minutes. Samples were electrophoresed at 18 mA constant current for 5hours at 4° C. using a Protein II xi cell power supply (BioRadLaboratories, Richmond, Calif., model 1000/500). The molecular weight ofeach individual protein as visually seen in the SDS-PAGE gel wasestimated using a GS-800 densitometer (BioRad) using a broad rangemolecular weight marker as a reference standard (BioRad).

The SDS-PAGE patterns of the proteins from each isolate when grown inthe presence of 1600 μM dipyridyl showed a very different proteinexpression pattern compared to the same strain when grown in thepresence of 300 μM dipyridyl. For instance, when grown in 300 μMdipyridyl isolate SAAV1 resulted in metal-regulated proteins of 90 kDa,84 kDa, 72 kDa, 66 kDa, 36 kDa, 32 kDa, and 22 kDa, while growth in 1600μM dipyridyl resulted in metal-regulated proteins of 87.73 kDa, 54.53kDa, 38.42 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, and 33.0 kDa. Likewise,when grown in 300 μM dipyridyl isolate 19636 resulted in proteins of 42kDa and 36 kDa, while growth in 1600 μM dipyridyl resulted inmetal-regulated proteins of 87.73 kDa, 54.53 kDa, 38.42 kDa, 37.37 kDa,35.70 kDa, 34.91 kDa, and 33.0 kDa. All conditions, including growth iniron-replete media, resulted in the expression of the following proteinsthat were presumably not metal-regulated: 150 kDa, 132 kDa, 120 kDa, 75kDa, 58 kDa, 50 kDa, 44 kDa 43 kDa 41 kDa, and 40 kDa.

Furthermore, growth of the different strains of S. aureus in 1600 μMdipyridyl resulted in similar protein expression patterns. Thecompositions enriched in iron-regulated membrane proteins from the avianisolate (SAAV1) included proteins with molecular weights of 87.73 kDa,54.53 kDa, 38.42 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, and 33.0 kDa. Themolecular weights of the proteins from the ATCC isolate 19636 wereessentially identical to those from the avian isolate. Both bovineisolates, when grown with 1600 μM 2,2-dipyridyl, expressed similarbanding profiles as the avian and ATCC isolates for the majority of theproteins (87.73 kDa, 54.53 kDa, 37.7 kDa, 35.70 kDa, 34.91 kDa, and 33.0kDa). However, neither of the bovine isolates produced the 38.42 kDaprotein seen with the avian and ATCC isolates, and the bovine isolatesexpressed three proteins (80.46 kDa, 65.08 kDa, and 31.83 kDa) notobserved with the avian and ATCC strains (see FIG. 1 and Table 7). Allconditions resulted in the expression of the following proteins thatwere not metal-regulated: 150 kDa, 132 kDa, 120 kDa, 75 kDa, 58 kDa, 50kDa, 44 kDa, 43 kDa, 41 kDa, and 40 kDa.

TABLE 7 Molecular weights of metal-regulated polypeptides obtained fromStaphylococcus aureus isolates. Avian Human Bovine Bovine SAAV1 196361477 2176 87.73 87.73 87.73 87.73 — — 80.46 80.46 — — 65.08 65.08 54.5354.53 54.53 54.53 38.42 38.42 — — 37.37 37.37 37.37 37.37 35.70 35.7035.70 35.70 34.91 34.91 34.91 34.91 33.0  33.0  33.0  33.0  31.83 31.83

Interestingly, there was no difference in the protein profiles asexamined by SDS-PAGE between the clarified supernatant and the bacterialpellet after treating the bacteria with lysostaphin/lysozyme. Both theextracted bacterial pellet and the supernatant had exactly the sameprotein profiles as examined by SDS-PAGE. This same observation was alsoseen when disrupting the bacterial cells using an AVESTIN homogenizer at30,000 psi. The resultant bacterial pellet, after slow speedcentrifugation was identical in its protein profile as compared to theclarified supernatant after high speed centrifugation at 30,000×g for2.0 hours at 4° C.

Example 2 Preparation of the Immunizing Compositions Derived fromStaphylococcus aureus

The proteins from the human isolate ATCC 19636 and the bovine isolate1477, grown in iron-deplete conditions and prepared as described inExample 1, were used to formulate two vaccine compositions. The proteinsfrom the ATCC isolate had molecular weights of 87.73 kDa, 54.53 kDa,38.42 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, and 33.0 kDa, while thebovine isolate expressed proteins having molecular weights 87.73 kDa,80.46 kDa, 65.08 kDa, 54.53 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, 33.0kDa, and 31.83. Each composition also contained the following proteinsthat were not metal-regulated: 150 kDa, 132 kDa, 120 kDa, 75 kDa, 58kDa, 50 kDa, 44 kDa, 43 kDa, 41 kDa, and 40 kDa. Stock vaccines wereprepared from the two strains by emulsifying each aqueous proteinsuspension (500 μg total protein/ml) into a commercial adjuvant(EMULSIGEN, MVP Laboratories, Ralston, Nebr.) using an IKA Ultra TurraxT-50 homogenizing vessel (IKA, Cincinnati, Ohio) to give a final dose of50 μg total protein in a 0.1 ml injectable volume with an adjuvantconcentration of 22.5% vol/vol. As a control vaccination, a proteincomposition was prepared from the bovine isolate 1477 grown underiron-replete conditions (TSB supplemented with 300 μM ferric chloride)as described in Example 1. A placebo vaccine was prepared bysubstituting physiological saline for the aqueous protein suspension inthe above protocol.

Example 3 Mouse Vaccination

Seventy (N=70) female CF-1 mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams were equallydistributed into 7 groups (10 mice/group). Mice were housed inpolycarbonate mouse cages (Ancore Corporation, Bellmore, N.Y.). A singlecage was used for each treatment group and food and water was suppliedad libitum to all mice. All mice were vaccinated intraperitoneally with0.1 ml of the appropriate composition two times at 14 day intervals asfollows:

Group-1: Placebo-Vaccinated

Group-2: Vaccinated with ATCC 19636 proteins expressed underiron-restriction.

Group-3: Placebo-Vaccinated

Group-4: Vaccinated with Bovine 1477 proteins expressed underiron-restriction.

Group-5: Vaccinated with Bovine 1477 proteins expressed underiron-restriction.

Group-6: Vaccinated with ATCC 19636 proteins expressed underiron-restriction.

Group-7: Bovine 1477 FeCl₃-Vaccinated, where “Bovine 1477 FeCl₃” refersto proteins obtained from Bovine 1477 grown in TSB supplemented with 300μM ferric chloride.

Example 4 Preparation of Challenge Organism

The previously described Staphylococcus aureus strains ATCC 19636 andstrain 1477 were used as challenge organisms. Briefly, the isolates fromfrozen stocks (previously described) were streaked onto blood agarplates and incubated at 37° C. for 18 hours. A single colony of eachisolate was subcultured into 50 ml Tryptic Soy Broth (Difco) containing1600 μM 2,2′ dipyridyl. The cultures were incubated at 37° C. for 6hours while rotating at 200 rpm, then centrifuged at 10,000×g for 10minutes at 4° C. to pellet the bacteria. The bacterial pellets werewashed twice by centrifugation in TBS at 4° C. The final pellets wereresuspended in TB S to an optical density of 42% Transmittance (T) at562 nm in a volume of approximately 25 ml of TBS and used for challenge.Just prior to challenge, 1 ml of these bacterial suspensions wasserially diluted and plated on agar to enumerate the number ofcolony-forming units (CFU) per mouse dose.

Example 5 Challenge

Fourteen days after the second vaccination, mice in all groups (1-7)were subcutaneously challenged in the back of the neck with 0.1 ml ofthe appropriate organism. The seven groups of mice were challenged asfollows:

Group-1 (Placebo-Vaccinated): Challenged with ATCC 19636

Group-2 (Vaccinated with ATCC 19636 proteins expressed underiron-restriction): Challenged with ATCC 19636

Group-3 (Placebo-Vaccinated): Challenged with Bovine 1477

Group-4 (Vaccinated Bovine 1477 proteins expressed underiron-restriction): Challenged with Bovine 1477

Group-5 (Vaccinated Bovine 1477 proteins expressed underiron-restriction): Challenged with ATCC 19636

Group-6 (Vaccinated ATCC 19636 proteins expressed underiron-restriction): Challenged with Bovine 1477

Group-7 (Bovine 1477 FeCl₃-Vaccinated): Challenged with Bovine 1477

As determined by the enumeration protocol described in Example 4, theconcentration of S. aureus 19636 used for challenge was 1.35×10⁸ CFU permouse dose, and the concentration of S. aureus 1477 used for challengewas 1.65×10⁸ colony CFU per mouse dose. Morbidity, mortality and grosspathology were recorded daily for 7 days after challenge.

When comparing the mice challenged with the ATCC 19636 isolate, 70% ofthe placebo-vaccinated Group 1 mice died within 7 days of challenge(Table 8 and FIG. 2). This demonstrated that strain 19636 caused a highrate of mortality in mice at the dose level administered. In contrast tothe mice in Group 1, only 10% of the mice in Group 2 died within 7 dayspost-challenge. These results illustrated that the mice challenged withstrain 19636 were significantly protected by vaccination with the 19636composition (p=0.020, Fischer's Exact test). Furthermore, a Kaplan-Meieranalysis of the time-to-death data indicated that the vaccine affordedsignificant (p=0.0042, logrank test) protection against homologouschallenge (FIG. 3). In addition, only 20% of the mice in Group 5 diedwithin 7 days of challenge, indicating that the bovine 1477 compositionoffered significant protection against challenge with the ATCC 19636strain (p=0.015 logrank test for mortality). When the data was analyzedby a Kaplan-Meier survival curve and logrank test (FIG. 4), theprotection against mortality was determined to be significant (p=0.015logrank test for mortality), indicating that the vaccine compositionderived from strain 1477 provided heterologous protection againstchallenge with strain 19636.

TABLE 8 Mortality of Vaccinated and Non-Vaccinated Mice FollowingChallenge with Staphylococcus aureus (human ATCC isolate 19636 andbovine isolate 1477). Groups # Mice # Dead Percent mortality (%)Group-1* (Placebo, ATCC 10 7/10 70 19636 Chlg) Group-2* (ATCC 19636, 101/10 10 Homologous Chlg) Group-3* (Placebo, 10 2/10 20 Bovine 1477 Chlg)Group-4* (Bovine 1477, 10 1/10 10 Homologous Chlg) Group-5* (Bovine1477, 10 2/10 20 Heterologous Chlg) Group-6* (ATCC 19636, 10 0/10 0Heterologous Chlg) Group-7* (Bovine 1477 10 2/10 20 FeCl₃, Bovine 1477Chlg) *Group-1, (Placebo-Vaccinated/Challenged with ATCC 19636) *Group-2(Vaccinated with ATCC 19636 proteins expressed underiron-restriction/Challenged with ATCC 19636) *Group-3(Placebo-Vaccinated/Challenged with Bovine 1477) *Group-4 (Vaccinatedwith Bovine 1477 proteins expressed under iron-restriction/Challengedwith Bovine 1477) *Group-5 (Vaccinated with Bovine 1477 proteinsexpressed under iron-restriction/Challenged with ATCC 19636) *Group-6(Vaccinated with ATCC 19636 proteins expressed underiron-restriction/Challenged with Bovine 1477) *Group-7 (Bovine 1477FeCl₃ -Vaccinated/Challenged with Bovine 1477)

When comparing the mice challenged with the bovine 1477 isolate, only20% of the mice in the placebo-vaccinated group (Group 3) died within 7days of challenge. However, challenge with the bovine 1477 isolateelicited the development of necrotic skin lesions on 6 (75%) of thesurviving mice of Group 3. These lesions were measured and the averagesize of the lesions on the surviving mice was 18.5 mm (Table 9). Incontrast, 20% of the Group 4 mice died within 7 days of challenge, butonly three (38%) of the surviving mice developed lesions (averagediameter, 2.7 mm). These results indicate that the bovine 1477composition offered significant homologous protection againstdevelopment of lesions in the mice challenged with the bovine strain1477 (p=0.009, Student's t-test). In addition, vaccination with the ATCC19636 composition protected against challenge with strain 1477, since nomice died in Group 6 and only three (30%) of the mice developed skinlesions (average diameter, 3.7 mm). Taken together, the reducedmortality and/or lesion development in the mice in Groups 5 and 6demonstrate the significant cross-protective nature of the compositionsderived from strains 19636 and 1477 (p=0.012, Student's t-test based onlesion size). In demonstration of the efficacy of the composition ascompared to the non-iron regulated proteins, 20% of the mice in Group 7died and 4 of the survivors developed skin lesions (average diameter,15.8 mm). The mice of Group 7 demonstrated some degree of protection byvaccination with the proteins of the 1477 isolate since fewer micedeveloped lesions compared to the placebo-vaccinated Group 3. However,the skin lesions observed on the mice in group 7 were more frequent andof a larger diameter than the lesions on the mice of Group 4, indicatingthat, relative to proteins isolated from cells grown under iron-repleteconditions, the proteins isolated from bacteria grown under ironrestriction offered superior protection against an identical challenge.

The cross-protective nature of the proteins observed in the mousechallenge study is supported by the similar molecular weights of theproteins from the S. aureus strains described in Example 1 (FIG. 1).Although there were noticeable differences in the SDS-PAGE profile ofthe proteins from the bovine-derived isolates, specifically the absenceof a 38.4 kDa protein and the presence of 3 additional proteins, theproteins from both strains 1477 and ATCC 19636 elicited heterologousprotection. These results indicate that the similar proteins betweenstrains 19636 and 1477 are likely responsible for the cross-protectionobserved in Groups 5 and 6. By contrast, the protein profiles fromstrain 1477 grown under iron-deplete and iron-replete conditions areobservably different. Those proteins isolated under iron-depletedconditions are more protective when compared to the proteins isolatedunder iron-replete conditions, demonstrated by the reduction in lesiondevelopment among the mice of Group 4 compared to the mice of Group 7.

TABLE 9 The Induction of Necrotic Lesions in Mice Seven DaysPost-Challenge with Staphylococcus aureus (ATCC Isolate 19636 and/orBovine Isolate 1477) Group-1 Group-2 Group-3 Group-4 Group-5 Group-6Group-7 Lesion diameter (millimeter) per mouse No lesion No lesion 26 55 5 25 No lesion No lesion 25 2 No lesion 5 25 No lesion No lesion 24 1No lesion 1 10 Dead No lesion 24 No lesion No lesion No lesion 3 Dead Nolesion 7 No lesion No lesion No lesion No lesion Dead No lesion 5 Nolesion No lesion No lesion No lesion Dead No lesion No lesion No lesionNo lesion No lesion No lesion Dead No lesion No lesion No lesion Nolesion No lesion No lesion Dead No lesion Dead No lesion Dead No lesionDead Dead Dead Dead Dead Dead No lesion Dead Average lesion diameter(mm) among surviving mice 0 0 18.5 2.7 5 3.7 15.8 *Group-1, (Placebo-Vaccinated/Challenged ATCC 19636) *Group-2 (Vaccinated with ATCC 19636proteins expressed under iron-restriction/Challenged ATCC 19636)*Group-3 (Placebo -Vaccinated/Challenged Bovine 1477) *Group-4(Vaccinated with Bovine 1477 proteins expressed underiron-restriction/Challenged Bovine 1477) *Group-5 (Vaccinated withBovine 1477 proteins expressed under iron-restriction/Challenged ATCC19636) *Group-6 (Vaccinated with ATCC 19636 proteins expressed underiron-restriction/Challenged Bovine 1477) *Group-7 (Bovine 1477 FeCl₃Vaccinated/Challenged Bovine 1477

Example 6

In mammals, it has been shown that the response to tissue injury orbacterial infection results in an acute inflammatory response. Thisresponse increases capillary permeability and phagocytic infiltrationresulting in the clinical signs recognized as inflammation; swelling,fever, pain and redness; if left uncontrolled, this may lead to death.The activation of humoral factors and the release of cytokines mediatesystemic events collectively known as the acute phase protein responsewhich results in a cascade of physiological and biochemical events. Theduration of this response is directly related to the severity of theinjury and magnitude of the systemic infection. It has beenwell-documented that during bacterial sepsis, major surgery, burns andother bodily trauma there is an alteration in the concentration of anumber of metal ions in serum such as, iron, copper, and zinc. Forinstance, during the acute phase of an infection there is a decrease inplasma levels of iron and zinc and an increase in copper. The alterationof these trace metal ions in serum may directly affect the severity orprogression of any bacterial infection.

In this study we examined the expression of proteins of Staphylococcusaureus under various conditions of metal ion restriction in order tomimic the expression of novel proteins that may be expressed duringsystemic invasion. The Staphylococcus aureus strains evaluated in thisstudy originated from clinical samples of three different species ofanimal; avian (strain SAAV1), human (strain 19636), and bovine (strains1477 and 2176). Briefly, cultures of each isolate were prepared frommaster seed stocks in 200 ml of Tryptic Soy Broth (TSB). Each culturewas grown while stirring at 200 rpm for 6 hours at 37° C. Ten ml of eachculture were transferred into 500 ml of deplete TSB containing one offour metal ion chelators; 2, 2-dipyridyl (Dp), 2-pyridylmethyl-ethylenediamine (TPEN), catechin, and naringenin (all obtained from Sigma, St.Louis, Mo.). In addition each culture was also grown in cation-repletemedia containing ferric chloride, zinc chloride and/or copper chlorideprepared at 300 μM concentrations. The metal ion chelators were used atthe following concentration; 2,2-dipyridyl (800 μM), catechin andnaringenin were used at 300 μM, and 2-pyridylmethyl-ethylene diamine wasused at a concentration of 100 μM. Cultures were grown with eachchelator for 8 hours, at which point the culture was subcultured asecond time for an additional 12 hours. Each culture was subcultured forthree consecutive passes at 12-hour intervals. At the end of the thirdpass, each culture was harvested by centrifugation at 10,000×g for 20minutes. Each culture was washed twice by centrifugation at 10,000×g andresuspended in 20 ml Tris-buffered saline, pH 7.2 at 4° C.

Each bacterial pellet was resuspended in 45 ml of Tris-buffered saline,pH 7.2 (25 mM Tris and 150 mM NaCl) and the resulting bacterialsuspensions were dispensed as 9-ml aliquots into 5 individual tubes,twenty tubes total. One milliliter of TBS containing 50 units oflysostaphin (Sigma, St. Louis, Mo.) was added to each tube to give afinal concentration of 5 units/ml. Following incubation at 37° C. for 30minutes while shaking at 200 rpm, 1 ml of TBS containing 0.1 mg oflysozyme (Sigma) was added to each tube. The bacterial suspensions werethen incubated for an additional 45 minutes while shaking at 200 rpm.Next, suspensions were centrifuged at 3050×g for 12 minutes at 4° C. topellet large cellular debris. The supernatants were collected byaspiration without disturbing the pellet. The supernatant was thencentrifuged at 39,000×g for 2.5 hours. The resulting pellets, enrichedfor metal-regulated membrane proteins, were resuspended in 200 μL Trisbuffer, pH 7.2. The protein solutions for each isolate were combined fora total volume of 1 ml and stored at −90° C.

The proteins obtained from the SAAV1, 19636, 1477 and 2176 S. aureusisolates grown under iron, zinc and copper deplete conditions includedmetal-regulated polypeptides.

Cell extracts, derived from each isolate were size-fractionated onSDS-PAGE gels using a 4% stacking gel and 10% resolving gel. Samples forelectrophoresis were prepared by combining 10 μl of sample with 30 μl ofSDS reducing sample buffer (62.5 mM Tris-HCL ph 6.8, 20% glycerol, 2%SDS, 5% beta-mercaptoethanol) boiled for 4 minutes. Samples wereelectrophoresed at 18 mA of constant current for 5 hours at 4° C. usinga Protein II xi cell power supply (BioRad Laboratories, Richmond,Calif., model 1000/500).

The SDS-PAGE patterns of the proteins grown under zinc and/or copperchelation showed unique banding patterns in all isolates that weredifferent when compared to the same isolates grown underiron-restriction in the presence of 2,2′-dyipyridyl. For example, whenthe 19636 isolate was grown under iron-restriction or in the presence ofthe chelator 2,2′-dyipyridyl, unique iron-regulated proteins wereexpressed at the 87.73 kDa, 54.53 kDa, 38.42 kDa, 37.37 kDa, 35.70 kDa,34.91 kDa and 33.0 kDa regions. These proteins were downregulated whenthe isolate was grown in the presence of ferric chloride. However, whenthe same isolate was grown in the presence of the zinc and or copperchelator, a novel subsets of proteins was expressed relative to theproteins expressed under iron-restriction; the new proteins havingmolecular weights of approximately 115 kDa, 88 kDa, 80 kDa, 71 kDa, 69kDa, 35 kDa, 30 kDa, 29, kDa and 27 kDa. In addition, an 87.73 kDaprotein was expressed under conditions of iron restriction orcopper-restriction but not when cultures were zinc-restricted. Theproteins expressed under iron-restriction appeared to be downregulatedwhen growth was under either zinc-restriction and/or copper-restriction,but not completely shut off as seen when the isolate was grown in ferricchloride.

It appears that there are novel proteins expressed when the organism isgrown under copper-restriction and/or zinc-restriction that are notexpressed when the same isolate is grown under iron-restrictedconditions. Since transitional metals are used by organisms to buildenzymes that catalyze various biochemical reactions, the metal ions mayplay a vital role in microbial survival during a systemic infection. Itis perhaps for this reason that during sepsis there is a transientdecrease in the availability of these transitional metals, making themunavailable for growth of the organism. These novel proteins could verywell enhance the protective efficacy of the existing composition grownunder iron-restriction because they may also be expressed by thebacteria under the metal ion restriction experienced during systemicinvasion.

Example 7 Compositions of the Present Invention can Also be ProducedUnder Large Scale Commercial Conditions Fermentation

A cryogenic vial of the working seed (2 ml at 10⁹ CFU/ml) as describedin Example 1 was used to inoculate 500 ml of Tryptic Soy Broth (TSB)without dextrose (Difco) pre-warmed to 37° C. containing 0.125 g/l2,2-dipyridyl (Sigma), 2.7 grams BiTek yeast extract (Difco) andglycerol (3% vol/vol). The culture was incubated at 37° C. for 12 hourswhile stirring at 200 rpm at which time it was used to inoculate 2liters of the above media and allowed to grow for an additional 4 hoursat 37° C. This culture was used to inoculate a 20-liter VIRTIS bench-topfermentor, (Virtis, Gardiner, N.Y.) charged with 13 liters of theabove-described media. The pH was held constant between 6.9 and 7.1 byautomatic titration with 50% NaOH and 10% HCL. The stirring speed wasadjusted at 400 rev/minute, and the culture aerated with 11 litersair/minute at 37° C. Foaming was controlled automatically by theaddition of 11 ml defoamer (Mazu DF 204 Chem/Serv, Minneapolis, Minn.).The culture was allowed to grow continuously at these conditions for 4hours at which time was sterilely pumped into a 150-liter fermentor (W.B. Moore, Easton, Pa.). The fermentor was charged with 120 literstryptic soy broth without dextrose (3,600.0 grams), BiTek yeast extract(600 grams), glycerol (3,600 ml), 2,2-dypyrdyl (3.0 grams) and Mazu DF204 defoamer (60 ml). The parameters of the fermentation were asfollows: dissolved oxygen (DO) was maintained at 30%+/−10% by increasingagitation to 220 rev/minute sparged with 60 liters of air/minute and 10pounds per square inch (psi) back pressure. The pH was held constantbetween 6.9 and 7.1 by automatic titration with 50% NaOH and 10% HCL andthe temperature maintained at 37° C. At hour 4.5 (OD₅₄₀ 8-9) of thefermentation the culture was transferred to a 1,500 liter New BrunswickScientific fermentor IF-15000 charged with 1200 liters tryptic soy brothwithout dextrose (36,000 grams), BiTek yeast extract (6,000 grams),glycerol (36,000 ml), 2,2-dypyrdyl (30.0 grams) and Mazu DF 204 defoamer(600 ml). The parameters of the fermentation were as follows: dissolvedoxygen (DO) was maintained at 60%+/−10% with supplemental oxygen byincreasing agitation to 300 rev/minute sparged with 300 to 1100 litersof air/minute and 5 pounds per square inch (psi) back pressure. Asfermentation progressed supplemental oxygen was added from 0-90liters/minute to assist in the control of dissolved oxygen. The pH washeld constant between 6.9 and 7.4 by automatic titration with 50% NaOHand 10% HCL and the temperature was maintained at 37° C.

At approximately 5 hours post inoculation of the large fermentor theculture was supplemented with additional nutrients by feeding 70 litersof media containing 18,000 grams TSB without dextrose, 3,000 grams yeastextract 30.0 grams 2,2-dipyridyl and 18,000 ml of glycerol. The rate offeed was adjusted to approximately 28 liters/hour while increasingagitation. At the end of the feed the fermentation was allowed tocontinue for an additional 4 hours at which point the fermentation wasterminated by lowing the temperature of the fermentor to 18° C. (OD₅₄₀35-40 at a 1:100 dilution).

Harvest

The bacterial fermentation was concentrated and washed using a PallFiltron Tangential Flow Maxiset-25 (Pall Filtron Corporation, Northboro,Mass.) equipped with three 30 ft² Alpha 300-K open channel filters,catalog No. AS300C5, (Pall Filtron) connected to a Waukesha Model U-60feed pump (Waukesha Cherry-Burrell, Delevan, Wis.) The original culturevolume of 1250 liters was reduced to 50 liters (2.5 liters/minute) usinga filter inlet pressure of 30 psi and a retentate pressure of 5-6 psi.The bacterial retentate was adjusted back up to 150 liters usingTris-buffered Saline pH 8.5 and then concentrated again to 50 liters tohelp remove any contaminating exogenous proteins, such as exoproteins toinclude secreted toxins and proteases. The elevated pH of thetris-buffered saline helps prevent much of the proteolytic degradationthat can occur during storage of the whole cell suspension. Proteaseinhibitors may be used instead of, or in addition to, an elevated pH.The retentate was mixed thoroughly while in the 200-liter tank using abottom mount magnetically driven mixer. The retentate was sterilelydispensed (3.5 liters) into sterile 4 liter Nalgene containers No. 2122and placed into a −20° C. freezer for storage as a breaking point in themanufacture, or could be further processed. The pellet mass wascalculated by centrifuging 30 ml samples of the fermented culture andfinal harvest. Briefly, pre-weighted 50 ml Nalgene conical tubes werecentrifuged at 39,000×g for 90 minutes in a Beckman J2-21 centrifugeusing a JA-21 rotor (Beckman Instruments, Palo Alto Calif.). At the endof the run, the supernate was poured off and the tubes were weighedagain. The pellet mass was calculated for each stage. The fermentationprocess yielded a wet pellet mass of approximately 60 kilograms.

Disruption

Eighty kilograms of bacterial cell slurry in Tris-buffered Saline pH 8.5was aseptically transferred into a steam in place 1000 liter jacketedprocess tank (Lee, Model 259LU) with a top mounted mixer (Eastern, ModelTME-1/2, EMI Incorporated, Clinton, Conn.) containing 900 liters TBS pH8.5. The bulk bacterial suspension was chilled to 4° C. with continuousmixing for 18 hours at 200 rpm at which time was disrupted byhomogenization. Briefly, the 1000 liter tank containing the bacterialsuspension was connected to a model C-500-B AVESTIN homogenizer,(Avestin Inc, Ottawa Canda). A second 1000 liter jacketed process tank(empty) was connected to the homogenizer such that the fluid in theprocess tank could be passed through the homogenizer, into the emptytank and back again, allowing for multiple homogenizing passes whilestill maintaining a closed system. The temperature during homogenizationwas kept at 4° C. At the start of the first pass, fluid was circulatedat 70 psi via a Waukesha model 10DO pump (Waukesha) through thehomogenizer (500 gallons/hour), while the homogenizer pressure wasadjusted to 30,000 psi. Prior to the first pass, two pre-homogenizingsamples were withdrawn from the homogenizer to establish a baseline fordetermining the degree of disruption and monitoring of pH. The degree ofdisruption was monitored by transmittance (% T at 540 nm at 1:100dilution) compared to the non-homogenized sample. The number of passesthrough the homogenizer was standardized to give a final percenttransmittance between 78-91% T at a 1:100 dilution preferably between86-91%. After homogenization, the tank was removed from the homogenizerand put onto a chiller loop at 4° C. and mixed at 240 rpm.

Protein Harvest

The disrupted bacterial suspension containing the iron-regulatedproteins as illustrated in FIG. 1 were collected by centrifugation usingT-1 Sharples, (Alfa Laval Seperations, Warminster, Pa.). Briefly, the1000 liter jacketed process tank containing the disrupted bacterialhomogenate was fed into 12 Sharples with a feed rate of 250 ml/minute at17 psi at a centrifugal force of 60,000×g. The effluent was collectedinto a second 1000 liter jacketed process tank through a closed sterileloop allowing for multiple passes through the centrifuges whilemaintaining a closed system. The temperature during centrifugation waskept at 4° C. The homogentae was passed 8 times across the centrifuges.Approximately 50% of the protein was collected after the second pass, atwhich point, the homogenate fluid was concentrated to ⅓ of its originalvolume, which shortened the process time for the next 6 passes. Thehomogenate tank was aseptically disconnected from the centrifuges andconnected to a Millipore Pellicon Tangential Flow Filter assembly(Millipore Corporation, Bedford, Mass.), equipped with a 25 ft²screen-channel series Alpha 30K Centrasette filter (Pall Filtron)connected to a Waukesha Model U30 feed pump for concentration. Afterconcentration, centrifugation was continued until the process wascompleted. Protein was collected after each pass. The protein wascollected, resuspended and dispensed in 50 liters Tris-buffered salinepH 8.5 containing 0.15% formulin (Sigma) as preservative.

Diafiltration

The protein suspension was washed by diafiltration at 4° C. to removeany exogenous proteins (proteases, toxins, cytoplasmic and metabolicenzymes etc). Briefly, the 50 liters of protein was sterilelytransferred into a 200 liter process tank containing 150 liters sterileTris-buffer saline, pH 8.5 equipped with a bottom mount Dayton mixer,Model 2Z846 (Dayton Electric, Chicago, Ill.) rotating at 125 rev/minute.The process tank was sterilely connected to a Millipore PelliconTangential Flow Filter assembly (Millipore Corporation), equipped with a25 ft² screen-channel series Alpha 30K Centrasette filter (Pall Filtron)connected to a Waukesha Model U30 feed pump. The 200 liter proteinsolution was concentrated by filtration to a target volume 50 liters atwhich point 150 liters of sterile saline was added. The proteinsuspension was then concentrated to approximately 50 liters. The proteinconcentrate was stored in a 50 liter jacketed process tank equipped witha top mounted mixer and stored at 4° C.

It is interesting to note that the composition derived from the largescale process using homogenization as a means of disruption generatedidentical banding profiles as examined by SDS-PAGE as compared to thesmaller scale process described in Example 1. These results show thatlysostaphin could be replaced as the bacterial lysis agent using theAVESTIN homogenizer C500-B. This discovery allows for the low costgeneration of large volumes of iron-regulated proteins fromstaphlylococci.

Example 8 Hyper-Immunization of Mice and Preparation of PolyclonalAntibody

Passive immunization with purified antibody isolated from micevaccinated with proteins derived from S. aureus strain ATCC 19636 grownunder iron-limiting conditions was protective against a homologous andheterologous S. aureus challenge. Fifteen adult CD1 mice were vaccinatedas described in Example 3 with the protein composition derived from S.aureus strain ATCC 19636 grown under iron-deplete conditions asdescribed in Examples 1 and 2. Mice were vaccinated intraperitoneally 3times at 7 day intervals with 50 μg of protein composition at eachvaccination. Seven days after the third immunization, mice were bledcompletely by cardiac puncture. Serum was pooled and antibody purifiedusing standard ammonium sulfate precipitation. Exogenous serum proteinswere removed first prior to antibody precipitation by adding 0.5 volumesof saturated ammonium sulfate pH 7.2. The solution was stirred at 100rpm for 24 hours at 4° C. The solution was again centrifuged at 3000×gfor 30 minutes. The supernatant was collected and precipitated again byadding enough saturated ammonium sulfate to bring the finalconcentration to 55% saturation. The solution was stirred at 100 rpm for24 hours at 4° C. The precipitate was centrifuged at 3000×g for 30minutes. The final pellet from each sample was resuspended into 2 ml PBSpH 7.2. The precipitated antibodies were then dialyzed using a 50,000molecular cut off dialysis tubing (Pierce, Rockford Ill.) for 30 hoursagainst three 1 liter changes of phosphate-buffered saline to removeammonium sulfate. The first two liter changes were preserved with 0.02%sodium azide. The final 1 liter buffer change contained no preservative.The dialysate was collected and centrifuged again to remove anyremaining debris at 3000×g for 30 minutes. The antibody solution wasstored at 4° C. for less then 48 hours prior to use. Each sample wasplated on blood agar to verify sterility prior to infusion.

Example 9 Passive Immunization and Challenge

In order to evaluate the protective effect of infused antibody raisedagainst S. aureus proteins expressed during iron-limitation, two groupsof 15 mice each were infused intraperitoneally with either the purifiedantibody preparation (Group 1) or physiological saline (Group 2) in a200 μL infusion. An additional two groups of 15 mice each were infusedsubcutaneously with either the purified antibody preparation (Group 3)or physiological saline (Group 4). After 60 minutes, the 2 groups of 15mice receiving an intraperitoneal infusion were challengedintraperitoneally with 1.3×10⁸ cfu of S. aureus strain 19636. Similarly,the 2 groups of 15 mice receiving a subcutaneous infusion werechallenged subcutaneously with 1.3×10⁸ cfu of S. aureus strain 1477 totest for cross-protection against challenge by a different S. aureusstrain. Mortality and/or lesion size was recorded for 5 days and thelivers of all mice were removed post-mortem, homogenized and plated todetermine the number of S. aureus present as a measure of systemicinfection. The Kaplan-Meier survival curves (FIGS. 5 and 6) show theprotective effect afforded by the infusion of antibodies from micevaccinated with the S. aureus proteins expressed during ironrestriction. Although the difference between the infused and controlgroups for the ATCC 19636-challenge groups was not significant (p=0.076,log-rank test), the liver of the single mouse that died within theantibody-infused group at Day 1 was cultured on blood agar to determinethe absence and/or presence of the challenge organism (S. aureus). Theculture derived from this mouse was negative for Staphylococcus andshowed no growth on the blood agar plate or culture medium. In contrast,the livers of the mice that died within the placebo group, were allpositive for the presence of Staphylococcus, in fact, pure cultures wereobtained on each blood agar plate derived from the livers of these mice.While the liver data do not preclude the possibility that the mouse thatdied within the antibody-infused group died of S. aureus infection, theinfection was not systemic, as it was in the placebo group, and themouse may have died for other reasons. Censoring of thisantibody-infused mouse death results in a significant difference betweenantibody-infused and placebo treatments (p=0.015, log-rank test). Thedata for the cross-challenge, where mice were infused with antibodygenerated after vaccination with ATCC 19636-derived proteins andchallenged by S. aureus strain 1477, also showed a protective trend.Between 7 and 14 days post challenge, all mice in the infused andnon-infused groups began to develop necrotic skin lesions. However,gross examination of mice clearly revealed a visible delay in theformation of an observable lesion as well as the severity of the lesionbetween the groups. Infused mice developed lesions more slowly ascompared to non-infused control mice which developed lesion faster theninfused mice and at a greater degree of severity. The infused micehealed faster then non-infused mice. This was clearly evident between 21and 35 days post challenge. Gross examination of mice at 35 days postchallenge showed that non-infused mice were severely disfigured andrevealed a greater degree of scarring. In fact, many of these mice lostnormal posture, in that they appeared twisted in appearance, in contrastto infused mice that did not develop nearly the extensive scar tissueand/or disfigurement as illustrated by the twisted appearance that thenon-infused mice developed. Overall, these data suggest thatinterperitoneal infusion of antibodies raised against S. aureusiron-induced proteins can both protect against and limit the severity ofS. aureus infection.

Example 10 Evaluation of a Vaccine Composition Derived fromStaphylococcus aureus in a Chronically Infected Dairy Herd

A commercial Dairy herd having a history of chronically high somaticcell counts attributable to Staphylococcus aureus was chosen for theevaluation of a vaccine composition as described in Example 1. Thecriterion for establishing vaccine efficacy of this experimental studywas: 1) decreased incidence of clinical mastitis caused byStaphylococcus aureus among vaccinates compared to non-vaccinatedcontrols, 2) improvement (i.e., a decrease) in somatic cell count amongvaccinates compared to controls and 3) decrease in culture positiveisolation rates of S. aureus between vaccinated and non-vaccinatedcontrols. Blood will be taken at the time of the first vaccination (day0) and again at 3 and 6 weeks post initial immunization. Injection sitereactions or systemic reactions following vaccinations were monitoredthroughout the study. In addition, bulk tank milk samples were culturedand quantitatively enumerated to determine if there was a decrease inthe number of CFU of Staphylococcus aureus cultured after vaccination.

Three of the Staphylococcus isolates derived from the chronicallyinfected lactating cows within the herd were grown under conditions ofiron-restriction and non-iron restricted conditions as described inExample 1. The three isolates were designated TTX101, TTX102, andTTX103. Extracted samples were examined by SDS-PAGE to compare bandingprofiles between isolates. Identical banding profiles were observedamong isolates examined; the compositions made from each isolateincluded proteins having molecular weights of 87.73 kDa, 80.46 kDa,65.08 kDa, 54.53 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, 33.0 kDa and31.83 kDa. These proteins are the same molecular weights as previouslydescribed in Table 7. In addition, when comparing the isolates identicalbanding profiles were seen with those proteins that were expressed inall conditions that were not regulated by iron: 150 kDa, 132 kDa, 120kDa, 75 kDa, 58 kDa, 50 kDa, 44 kDa, 43 kDa, 41 kDa, and 40 kDa. Theseresults were consistent with previous observations. One isolatedesignated as TTX101 was chosen as the isolate to manufacture acomposition to be used in this study.

Example 11 Vaccine Preparation of Staphylococcus aureus (TTX101)

A composition was prepared as described in Example 1 using the isolateTTX101. The composition included proteins expressed under iron depleteconditions having molecular weights of 87.73 kDa, 80.46 kDa, 65.08 kDa,54.53 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, 33.0 kDa, and 31.83 kDa aswell as non-metal-regulated proteins having molecular weights of 150kDa, 132 kDa, 120 kDa, 75 kDa, 58 kDa, 50 kDa, 44 kDa 43 kDa 41 kDa, and40 kDa. The immunizing composition derived from strain TTX101 was usedto prepare the experimental vaccine by emulsifying the extracted proteinsuspension (400 μg total protein per milliliter) into a commercialadjuvant (EMULSIGEN, MVP Laboratories, Ralston Nebr.) using an IKA UltraTurrax T-50 homogenizing vessel (IKA, Cincinnati, Ohio) to give a finaldose of 800 μg total protein in a 2.0 ml injectable volume with anadjuvant concentration of 22.5% vol/vol. The vaccine was administeredsubcutaneously 2 times at 21 day intervals.

Example 12 Experimental Design and Herd Vaccination

Eighteen days before the first vaccination all lactating cows enrolledin the study (N=80) were tested for S. aureus by standardized aerobicbacteriological culture methods by culturing individual milk samplesderived from each lactating cow. In addition, the Somatic Cell Counts(SCC) were enumerated by the Dairy Herd Improvement Association usingstandard methods. Fourteen of the 80 cows were clinically diagnosed withmastitis and were culture positive for S. aureus. The remaining cows(N=66) tested negative for S. aureus. The eighty cows were equallydivided into two groups designated as group-1, vaccinated (N=40) andgroup-2, non-vaccinated (N=40). The fourteen clinically diagnosedStaphylococcus positive cows were equally distributed between bothgroups so that each study group contained 7 cows with clinical mastitis.The average SCC between groups prior to the first vaccination was203,219 in the non-vaccinated controls compared to 240,443 in vaccinates(not statistically different p=0.7).

Eighteen days after the first sampling all cows in group 1 werevaccinated subcutaneously in the upper right shoulder with 2 ml ofvaccine as described in Example 11. Ten days after the first vaccinationmilk samples were taken at this time period by the DHIA for theenumeration of somatic cells from each individual cow. Milk samples werenot bacteriologically tested at this time period for determining thepresence of Staphylococcus. The difference in the SCC between groups atthis time period was 125,241 (vaccinates) compared to 196,297(controls). This was a 36% difference in the number of somatic cellsbetween vaccinates as compared to non-vaccinated controls. Thedifference in the SCC between the controls and vaccinates at thissampling period was not statistically different (p=0.5). The lack ofstatistical difference in the SCC between groups at both samplingperiods was due to the large variation in individual SCC between cows.The injection site of each vaccinated cow was also examined at this sametime period. None of the cows examined showed any adverse tissuereaction at the site of injection by physical examination. In addition,there was no measurable loss in milk production due to vaccination.

Twenty one days after the first vaccination all cows in group-1(vaccinates) were given their second vaccination or booster. During thetime period between first and second vaccination, cows in both groups(vaccinates and controls) developed teat damage due to a dramatic dropin the environmental temperature resulting in the formation of lesionsat the end of the teat, resulting in the development of infected teatsand potentially increasing the isolation of Staphylococcus duringsampling, which was observed at the third sampling period. Twenty threedays after the second vaccination milk samples were taken by the DHIAfor the enumeration of Somatic Cells from each individual cow. Milksamples were also bacteriologically tested for the presence ofStaphylococcus aureus. There was a dramatic increase in isolation rateof S. aureus at this time period in the cows that tested negative at thefirst sampling period. In the non-vaccinated controls 42.9% of thesecows now tested positive for S. aureus, in contrast to the vaccinates,which only showed and increase of 35.5%. This was a 7.4% differencebetween vaccinates as compared to the non vaccinated controls. It'sdifficult to say that the improvement in the isolation rate of S. aureusin the vaccinated group was due to the effect of the vaccine alone. Onecannot overlook the difficulty in obtaining clean milk samples from cowsthat had teat damage which could increase the potential contamination ofthe milk by S. aureus when obtaining the sample. Nevertheless, there wasa significant difference in the average SCC between vaccinates comparedto controls. The average SCC of the vaccinated group was 222,679compared to 404,278 somatic cells as measured in the control group. Thiswas a 44.9% difference between vaccinates when compared to the nonvaccinated controls. It's interesting to speculate that the differenceseen in the SCC between these groups also coincides with the differencein the isolation rate of S. aureus between groups. However, due to thelarge variation in SCC between individual animals and the small samplesize of the experimental trial in the number of animals the differencewas not statistically different (p=0.28).

At this same time period the injection site of each vaccinated cow wasexamined for any adverse tissue reaction that may have been caused bythe vaccine composition. None of the cows examined showed any adversereaction at the site of injection by physical examination. The vaccinecompositions appeared to be highly tissue compatible and caused nomeasurable loss in milk production after each vaccination.

Monitoring of the cows is continued by measuring SCC and milk samplesfor the presence or absence of Staphylococcus aureus. Some of the cowsof each group are vaccinated a third time at 42 days after the secondvaccination. There appears to be a difference favoring the use of thevaccine composition for decreasing somatic cell counts and controllinginfection caused by Staphylococcus aureus. Further monitoring includesserology based on antibody titers to the vaccine composition, changes inmilk production in vaccinated cows due the improvement in health, anddecreased SCC of vaccinated animals compared to non-vaccinated cohorts.In addition, other experiments are conducted to investigate theprotective index of the vaccine based on dose response followingchallenge with a virulent S. aureus.

Example 13

Since the molecular weights of the proteins among the different S.aureus strains have been demonstrated to be similar and sinceheterologous protection was observed in the mouse challenge study, wesought to determine if the proteins sharing similar molecular weights inFIG. 1 were similar proteins. The technique chosen to characterize theproteins was matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS). A portion of the composition was resolved usingSDS-PAGE as described in Example 1, and the gel was stained withCoomassie Brilliant blue to visualize the proteins.

Materials and Methods

Excision and washing. The gel was washed for 10 minutes with watertwice. Each protein band of interest was excised by cutting as close tothe protein band as possible to reduce the amount of gel present in thesample.

Each gel slice was cut into 1×1 mm cubes and placed in 1.5 ml tube. Thegel pieces were washed with water for 15 minutes. All the solventvolumes used in the wash steps were approximately equal to twice thevolume of the gel slice. The gel slice was next washed withwater/acetonitrile (1:1) for 15 minutes. When the proteins had beenstained with silver, the water/acetonitrile mixture was removed, the gelpieces dried in a SPEEDVAC vacuum concentrator/dryer (ThermoSavant,Holbrook, N.Y.) and then reduced and alkylated as described below. Whenthe gel pieces were not silver-stained, the water/acetonitrile mixturewas removed, and acetonitrile was added to cover until the gel piecesturned a sticky white, at which time the acetonitrile was removed. Thegel pieces were rehydrated in 100 mM NH₄HCO₃, and after 5 minutes, avolume of acetonitrile equal to twice the volume of the gel pieces wasadded. This was incubated for 15 minutes, the liquid removed, and thegel pieces dried in a SPEEDVAC.

Reduction & alkylation. The dried gel pieces were rehydrated in 10 mMDTT and 100 mM NH₄HCO₃, and incubated for 45 minutes at 56° C. Afterallowing the tubes to cool to room temperature, the liquid was removedand the same volume of a mixture of 55 mM iodoacetamide and 100 mMNH₄HCO₃ was immediately added. This was incubated for 30 minutes at roomtemperature in the dark. The liquid was removed, acetonitrile was addedto cover until the gel pieces turned a sticky white, at which time theacetonitrile was removed. The gel pieces were rehydrated in 100 mMNH₄HCO₃, and after 5 minutes, a volume of acetonitrile equal to twicethe volume of the gel pieces was added. This was incubated for 15minutes, the liquid removed, and the gel pieces dried in a Speed vac. Ifthe gel was stained with coomasie blue, and residual coomassie stillremained, the wash with 100 mM NH₄HCO₃/acetonitrile was repeated. In geldigestion. Gel pieces were completely dried down in a Speed

Vac. The pieces were rehydrated in digestion buffer (50 mM NH₄HCO₃, 5 mMCaCl₂, 12.5 nanograms per microliter (ng/μ1) trypsin) at 4° C. Enoughbuffer was added to cover the gel pieces, and more was added as needed.The gel pieces were incubated on ice for 45 minutes, and the supernatantremoved and replaced with 5-2 μl of same buffer without trypsin. Thiswas incubated at 37° C. overnight in an air incubator.

Extraction of peptides. A sufficient volume of 25 mM NH₄HCO₃ was addedto cover gel pieces, and incubated for 15 minutes (typically in a bathsonicator). The same volume of acetonitrile was added and incubated for15 minutes (in a bath sonicator if possible), and the supernatant wasrecovered. The extraction was repeated twice, using 5% formic acidinstead of NH₄HCO₃. A sufficient volume of 5% formic acid was added tocover gel pieces, and incubated for 15 minutes (typically in a bathsonicator). The same volume of acetonitrile was added and incubated for15 minutes (typically in a bath sonicator), and the supernatant wasrecovered. The extracts were pooled, and 10 mM DTT was added to a finalconcentration of 1 mM DTT. The sample was dried in a SPEEDVAC vacuumconcentrator/dryer to a final volume of approximately 5 μl.

Desalting of peptides. The samples were desalted using a ZIPTIP pipettetips (C18, Millipore, Billerica, Mass.) as suggested by themanufacturer. Briefly, a sample was reconstituted in reconstitutionsolution (5:95 acetonitrile:H₂O, 0.1%-0.5% trifluoroacetic acid),centrifuged, and the pH checked to verify that it was less than 3. AZIPTIP was hydrated by aspirating 10 μl of solution 1 (50:50acetonitrile:H₂O, 0.1% trifluoroacetic acid) and discarding theaspirated aliquots. This was followed by aspirating 10 μl of solution 2(0.1% trifluoroacetic acid in deionized H₂O) and discarding theaspirated aliquots. The sample was loaded into the tip by aspirating 10μl of the sample slowly into the tip, expelling it into the sample tube,and repeating this 5 to 6 times. Ten microliters of solution 2 wasaspirated into the tip, the solution discarded by expelling, and thisprocess was repeated 5-7 times to wash. The peptides were eluted byaspirating 2.5 μl of ice cold solution 3 (60:40, acetonitrile:H₂O, 0.1%trofluoroacetic acid), expelling, and then re-aspirating the samealiquot in and out of the tip 3 times. After the solution has beenexpelled from the tip, the tube is capped and stored on ice.

Mass spectrometric peptide mapping. The peptides were suspended in 10□μl to 30 μl of 5% formic acid, and analyzed by MALDI-TOF MS (BrukerDaltonics Inc., Billerica, Mass.). The mass spectrum of the peptidefragments was determined as suggested by the manufacturer. Briefly, asample containing the peptides resulting from a tryptic digest weremixed with matrix cyano-4-hydroxycinnamic acid, transferred to a target,and allowed to dry. The dried sample was placed in the massspectrometer, irradiated, and the time of flight of each ion detectedand used to determine a peptide mass fingerprint for each proteinpresent in the composition. Known polypeptides were used to standardizethe machine.

Data analysis. The experimentally observed masses for the peptides ineach mass spectrum were compared to the expected masses of proteinsusing the Peptide Mass Fingerprint search method of the Mascot searchengine (Matrix Science Ltd., London, UK, and www.matrixscience.com, seePerkins et al., Electrophoresis 20, 3551-3567 (1999)). The searchparameters included: database, MSDB or NCBInr; taxonomy, bacteria(eubacteria) or Firmicutes (gram-positive bacteria); type of search,peptide mass fingerprint; enzyme, trypsin; fixed modifications,carbamidomethyl (C) or none; variable modifications, oxidation (M),carbamidomethyl (C), the combination, or none; mass values,monoisotopic; protein mass, unrestricted; peptide mass tolerance,between ±150 ppm and ±430 ppm, or ±1 Da; peptide charge state, Mr; maxmissed cleavages, 0 or 1; number of queries, 20.

Results

The result of this search was a mass fingerprint for each proteinpresent in the composition is shown in Tables 2, 3, 4, and 5.

Example 14 Identification of Iron-Regulated Protein Families UsingMicroarray-Based Gene Expression Analysis of S. aureus Grown Under LowIron Conditions

For microarray analysis, bacteria were cultured in chemically definedmedia (CDM) made from individual stock solutions (Table 10).

TABLE 10 Chemically defined medium (CDM) for Staphylococcus aureus[Final] Stock composition Add to 1 L Salts (20X) g/L g/500 ml 50 mlK₂HPO₄ 7 70 KH₂PO₄ 2 20 Na₃citrate 1.47 14.7 (NH₄)₂SO₄ 1 10 Carbohydrate(40X) g/L g/500 ml 25 ml Glucose 5 100 Vitamins (1000X) mg/L mg/100 ml 1ml Thiamine 1 100 Nicotinic acid 0.5 50 Biotin 0.005 dilution* Calciumpantothenate 0.25 25 Nucleotides (100X) mg/L mg/100 ml* 10 ml *Dissolvein 100 ml 2N HCl Adenine 5 50 Guanine 5 50 Cytosine 5 50 Uracil 5 50Thymine 10 200 Micronutrients (1000X) μM mg/100 ml stockAB* 1 ml *Makestocks A and B, then add 1 ml of each to 98 ml ddH20 to make final stock(A) CaCl₂ 0.5 735 H₃BO₃ 0.5 309 CoCl₂ 0.05 118 (NH₄)₆Mo₇O₂₄ 0.005 62 (B)CuSO₄ 0.1 125 MnSO₄ 0.1 169 ZnSO₄ 0.05 144 Individual MgSO₄ 100 FeSO₄ orother 10-50 Amino acids (200X) mg/L g/100 ml 5 ml *Autoclave unlessotherwise noted Refrigerate Aspartic acid (0.1M HCl) 90 1.8 Proline 801.6 Alanine 60 1.2 Histidine 20 0.4 Valine 80 1.6 Arginine 50 1.0 Serine30 0.6 Methionine 3 0.06 Isoleucine 30 0.6 Dark refrigerated filteredTryptophan 10 0.2 Tyrosine (0.5M NaOH) 50 1.0 Room temperature Glutamicacid 100 2.0 Leucine 90 1.8 Phenylalanine 40 0.8 Glycine 50 1.0Threonine 30 0.6 Lysine 50 1.0 Fresh daily Cysteine 20 0.4

METHOD OF MAKING. For making iron-deplete media, combine all stocksolutions except cations and micronutrients and bring to proper volumein volumetric flask using MilliQ purified water and leaving enough voidvolume to accommodate cation additions. Add 15 g CHELEX resin per 1 Lmedia and stir at room temperature for at least 2 hours. Filter solutioninto sulfuric acid (10%) treated glass bottle using 2 μm bottle-topfilter. Add filtered cation stock solutions and store at 4° C. in thedark for up to 2 weeks.

S. aureus strains RF122 (isolated from bovine mastitis) and MSA553(isolated from human toxic shock syndrome) were used. Both isolates werestreaked on tryptic soy broth agar directly from secondary-passagefreezer stocks prior to use in experiments. CDM contained final citrateconcentrations approximately analogous to that in bovine milk (5 mM).For iron-free CDM, the following components were combined (0.998 L totalvolume) and added to 15 g CHELEX resin (BioRad Laboratories, Hercules,Calif.), then stirred for 1.5 hours at room temperature: salt, glucose,amino acids, vitamins, and nucleotides. The deferrated base media wasthen filtered using 2 μm bottle-top filters (Nalgene Nunc International,Rochester N.Y.) into sulfuric acid-treated bottles, after whichmicronutrients and 100 μM MgCl₂ (both solutions prepared in acid-treatedglassware with MilliQ water) were added. CDM was stored at 4° C. in thedark until use.

A single bacterial colony was inoculated into 3 ml iron-deplete CDM in a25 ml acid-treated glass culture tube and shaken overnight at 250 rpm ina 37° C. incubator. One milliliter of the subculture was then used toinoculate 500 ml of CDM in a 2500 ml Erlenmeyer flask the following day.Cultures were incubated at 37° C. with shaking at 250 rpm. Iron-depleteCDM cultures took approximately twice as long as CDM+50 μM FeSO₄ toreach an OD of 1.0 (18 hrs versus 36 hours). At mid-log phase(OD=0.600), 4×100 ml culture aliquots were distributed into 500 mlErlenmeyer flasks and allowed to shake in the incubator for 10 minutesprior to the addition of experimental iron solutions. To one flask, 300μl bovine lactoferrin (50 mg/ml, Sigma-Aldrich, St. Louis, Mo.) wereadded for a final concentration of 150 μg/ml. To another flask, 50 μlferric citrate (100 mM) were added. The remaining two control flasksreceived no supplements. At 5, 30, 60 and 120 minutes, 7.5 ml of culturewere collected and added to 5 ml guanidine thiocyanate solutioncontaining β-mercaptoethanol and 0.5% sodium lauryl sarcosine. Solutionswere mixed thoroughly to halt transcription and centrifuged at 4,000×gfor 8 minutes at 8° C.; supernatant was poured off and cells were frozenin 250 μl Trizol (Invitrogen) using an ethanol/dry ice bath, then storedat −80° C. until RNA extraction.

For RNA extraction, cell pellets were thawed on ice and 750 μl Trizol(Invitrogen, Carlsbad Calif.) were added. Cells were resuspended byvortex and the slurry was transferred to a 2 ml screw cap tubecontaining 0.1 mm silica-zirconium beads, then beat 3×2 minutes in aBeadBeater (Biospec Products, Inc., Bartlesville, Okla.) with iceincubation between repetitions. Slurries were incubated an additional 20minutes at room temperature, followed by centrifugation to pellet beadsand cellular components. 400 μl chloroform were added and mixed byinversion, incubated for 10 minutes at room temperature and tubes werecentrifuged 8 minutes at 12,000×g at 8° C. The aqueous layer was removedand the RNA precipitated with 400 μl isopropanol followed by washingwith 1 ml 70% ethanol. Clear RNA pellets were air-dried briefly andresuspended in 100 μl RNase-free H₂O. DNA was digested using standardDNase kit (Qiagen, Valencia Calif.) followed by cleanup according tomanufacturer's recommendations on RNeasy columns (Qiagen). Finally, aTurbo DNA-free kit (Ambion, Austin Tex.) was used to ensure eliminationof DNA from the preparation. RNA was measured on a spectrophotometer andrun on an Agilent Bioanalyzer (Agilent, Palo Alto Calif.) to verifyquality and quantity prior to the generation of cDNA for microarrayhybridization.

Microarray analysis was carried out according to established protocols.The array, featuring 3841 70 mer oligonucleotides (Illumina, San DiegoCalif.) representing open reading frames (ORFs) from nine sequenced S.aureus genomes including RF122 and MSA553, was spotted in triplicate onGaps II aminosaline coated slides (Corning, Acton Mass.) using aBioRobotics Microgrid II Array Spotter (BioRobotics, Cambridge UK).Slides were rehydrated, UV cross-linked and stored under dessication.Immediately prior to hybridization, slides were incubated for 1 hour at42° C. in prehybridization buffer consisting of 25 ml formamide, 12.5 ml20×SSC, 12 ml dH₂O, 500 μL 10% SDS and 0.5 g BSA. Slides were rinsedwith 2 L MilliQ water and dried by centrifugation. To prepare samples,8-10 μg total bacterial RNA were incubated with 20 μg random hexamers at70° C. for 10 minutes, followed by reverse transcription withamino-allyl incorporation using Superscript II (Invitrogen) andamino-allyl coupled dUTP (Sigma). Labeled cDNA was neutralized,purified, dried and resuspended with Cy3 or Cy5 fluorescent dyes(Amersham Biosciences Corp., Piscataway N.J.); coupling proceeded for 2hours. Fluorescently labeled cDNA samples (12 μl each) were washed usinga Qiagen PCR purification kit, combined and added to 9.8 μl formamide,6.8 μL 20×SSC, 3.4 μl salmon sperm DNA (10 mg/ml, Invitrogen) and 1 μl10% SDS. Samples were incubated for 2 minutes at 99.9° C. in a thermalcycler and allowed to cool prior to array application. Probes were thenapplied to array, covered with a glass coverslip and incubated overnightin a 42° C. waterbath. Slides were washed thoroughly in diluted SSCbuffers after 12-16 hours of incubation and scanned using an Axon 4100BScanner and Axon GenePix Software (Axon Instruments, Union City Calif.).Raw intensity data were exported to GeneSpring (Agilent Technologies,[Silicon Genetics], Palo Alto Calif.) for normalization and filtering.Spots were globally normalized, filtered based on minimum (>1500) rawintensity values and the triplicates were averaged. Each experiment wasrun twice and a single slide was run for each using a dye-swap betweenmatching timepoints. Thus, at least 6 dye-swapped datapoints weregenerated for each gene at each timepoint, representing at least 2biological replicates. Data were further analyzed by hierarchicalclustering (Euclidian distance, average linkage, UPGMA) and K-meansclustering (uncentered correlation based measured distance) usingEPCLUST (Jaak Vilo, EBI) and SpotFire (SpotFire, Somerville, Mass.).Significance Analysis for Microarrays (SAM, (157)) was used onmedian-centered log ratios using the one-class model across alltimepoints to determine if expression of the gene differed significantlyfrom zero. Stringent delta values were used so that the percentage offalse positives was estimated to be zero. A summary of operons showingsimilar up or downregulation by SAM analysis is shown in Table 11,supporting the ability of the arrays to detect biological responses.

TABLE 11 Operonic clusters with coordinated transcriptional responsesidentified using microarray analysis of gene expression of S. aureus No.of coordinately expressed probes Contiguous Functionally Operonunregulated related but not ID Function Response genes contiguous Sircation upregulated 3 0 transport in low iron Fhu cation upregulated 3 1transport in low iron Opp oligopeptide upregulated 9 N/A transport inlow iron Mnt cation upregulated 3 1 transport in low iron Pfl (Formatefermentation upregulated 2 4 acetyl- in presence transferase) oflactoferrin

For standard cloning of proteins, the appropriate genes were amplifiedfrom DNA extracted from S. aureus (strain ATCC19636) by standardpolymerase chain reaction. Primers were designed to incorporate StuI andKpnI restriction endonuclease sites and are shown below.

TABLE 12 Cloning Primers: Gene (primer) Primer Sequence SEQ ID NO Pflb(5′ to 3′) GCAGGCCTTTAGAAACAAATAAAAATCATG 507 Pflb (3′ to 5′)TATGGTACCTTACATACTTTCATGGAATGTACG 508 Opp1A (5′ to 3′)GCAGGCCTAAAAAAGAAAACAAGCAATTAA 509 Opp1A (3′ to 5′)TATGGTACCTTATTTATACTGCATTTCATTGAA 510 SirA (5′ to 3′)GCAGGCCTTCATCTGATAGCA AAGATAAGG 511 SirA (3′ to 5′)TATGGTACCTTATTTTGATTGTTTTTCAATATT 512 SYN2 (5′ to 3′)GCAGGCCTAAAGAATCATCAACTAAA 513 SYN2 (3′ to 5′)TATGGTACCCTTTTGTTCTTTTTTTGA 514 FhuD (5′ to 3′)GCAGGCCTACTGAAGAGAAAACTGAAATGA 515 FhuD (3′ to 5′)TATGGTACCTTATTTTGCTTTTTCTGCAATTTT 516 SYN1 (5′ to 3′)GCAGGCCTGGTAGCGACGATAATGGCTCGT 517 SYN1 (3′ to 5′)TATGGTACCTTATTTTCTATAAATTGCATCTCT 518 MntC (5′ to 3′)GCAGGCCTAGTGATAAGTCAAATGGCAAACTA 519 MntC (3′ to 5′)TATGGTACCTTATTTCATGCTTCCGTGTACAG 520 SstD (5′ to 3′)GCAGGCCTTCAGAAACTAAAGGTTCTAAAGAT 521 SstD (3′ to 5′)TATGGTACCTTATTTTACAACTTTTTCAAGTT 522 FhuD2 (5′ to 3′)GCAGGCCTACTAAATCTTATAAAATGGACGAT 523 FhuD2 (3′ to 5′)TATGGTACCTTATTTTGCAGCTTTAATTAATT 524

DNA extracted from S. aureus ATCC19636 was used as the template. DNAamplicons were verified by gel electrophoresis and the amplified bandsof DNA were excised, purified, digested and ligated into cut pQE30-Xavector, transformed into competent XL-1 E. coli and screened forampicillin resistance. Resistant clones were screened for plasmidinserts using colony PCR.

Example 15 Screening of Immunoreactivity of Protective ProteinCandidates

To evaluate the antibody reactivity of the proteins identified fromMALDI-TOF analysis (Example 13) and/or microarray and genomic analysis(Example 14), a two-part screen was used to evaluate individuallyexpressed staphylococcal proteins. The rapid first screen usedtranscriptionally active PCR fragments to survey antibody binding tosmall amounts of candidate protein expressed using a cell-free E. colilysate. The second screen used standard PCR-based cloning, expressionand purification of proteins in E. coli using a commercial vector(pQE30Xa, Qiagen, Valencia Calif.) in order to validate positivecandidates from the first screen. The second screen also generatedmaster seed stocks of E. coli host cells containing the expressionvector corresponding to each immunoreactive protein candidate forproduction and purification of sufficient protein for vaccination andexperimentation.

A high-throughput method for generating individual SIRP antigens wasused to test several candidate genes encoding S. aureus proteinsinvolved in metal metabolism. This method generates a transcriptionallyactive PCR (TAP) amplicon using a 2-step PCR reaction with primers thatadd a promoter, terminator and C-terminal His₆ tag. The resultingtranscriptionally active amplicons were used as a template for proteinproduction in a cell-free in vitro transcription/translation reactionconsisting of E. coli cell lysate, amino acids and buffers. The two-stepPCR reaction required a first set of primers specific to the gene ofinterest that also included a linker sequence matching the second set ofprimers. Each primer set for the first step of PCR was designed toexclude membrane-processing signal sequences to prevent integration intothe cell membrane and are shown below.

TABLE 13 TAP Primers: Gene (primer) Primer Sequence SEQ ID NO Pflb(5′ to 3′) AGAAGGAGATATACCATGTTAGAAACAAAT 525 Pflb (3′ to 5′)TTAATGATGATGATGATGATGCATACTTTCATG 526 Opp1A (5′ to 3′)AGAAGGAGATATACCATGAGAAAACTAACT 527 Opp1A (3′ to 5′)TTAATGATGATGATGATGATGTTTATACTGCAT 528 SirA (5′ to 3′)ATAAGGAGATATACCATGAATAAAGTAATT 529 SirA (3′ to 5′)TTAATGATGATGATGATGATGTTTTGATTGTTT 530 SYN2 (5′ to 3′)AGAAGGAGGATATACCATGAGAGGTCTAAAAACTTTT 531 SYN2 (3′ to 5′)TTAATGATGATGATGATGATGCTTTTGTTCTTTTTTTGA 532 FhuD (5′ to 3′)AGAAGGAGGATATACCATGAATAGGAATATCGTTAAA 533 FhuD (3′ to 5′)TTAATGATGATGATGATGATGTTTTGCTTTTTCTGCAAT 534 SYN1 (5′ to 3′)AGAAGGAGGATATACCATGAAGAAATCGTTAATTGCT 535 SYN1 (3′ to 5′)TTAATGATGATGATGATGATGTTTTCTATAAATTGCATC 536 MntC (5′ to 3′)AGAAGGAGATATACCAAAAAATTAGTA 537 MntC (3′ to 5′)TTAATGATGATGATGATGATGTTTCATGCTTCC 538 SstD (5′ to 3′)AGAAGGAGATATACCATGAAGAAAACAGTC 539 SstD (3′ to 5′)TTAATGATGATGATGATGATGTTTTACAACTTT 540 FhuD2 (5′ to 3′)AGAAGGAGATATACCATGAAAAAACTATTA 541 FhuD2 (3′ to 5′)TTAATGATGATGATGATGATGTTTTGCAGCTTT 542

A standard 50 μl PCR reaction was performed using (1 unit High FidelityTaq DNA polymerase (Invitrogen), 0.2 μM primers, 2 mM dNTP (each), 2 mMfinal Mg⁺⁺, and approximately 5 ng of starting DNA template, buffered to60 mM TrisSO₄ (pH 8.9) and 18 mM ammonium sulfate). The PCR cyclingprotocol included 1 minute of initial denaturation at 94° C., followedby 30 cycles as follows: Denature/94° C./30s; Anneal/55° C./30s;Extend/68° C., 90s. Identical primers with the appropriate overlap wereutilized for the second step PCR reaction and were supplied by themanufacturer (Genlantis). The resulting DNA PCR product was purified toeliminate residual primer, salt and DNA fragments and used as a templatefor a second reaction with a standard set of primers to add the promoterand terminator sequences using similar conditions. The DNA template wasthen purified and added to an E. coli cell-free Rapid Translation SystemRTS 100 reaction mix (Roche) containing 12 μl of E. coli lysate, 12 μlof amino acids, 10 μl of reaction mix, 1 μl of added methionine, 5 μl ofreconstitution buffer and 10 μl of purified DNA template from thetwo-step PCR reaction. Following incubation for 5 hours at 30° C., onemicroliter of each protein sample (approximately 0.5 μg/μl totalprotein) was applied to polyvinylidene fluoride (PVDF) membrane aftermethanol saturation. The blot was blocked overnight with 5% NFDM/TTBS,incubated with iron-restricted protein, enhanced (IRPE) hyperimmunizedmouse sera diluted 1:500 or anti-His₆ antibody (1:500), washed,incubated with secondary goat-anti mouse alkaline phosphatase (AP)conjugate (1:3000), washed and developed chromogenically (Bio-Rad APcolor development kit) for 20 minutes. Lysates containing seroreactivepolypeptides were identified.

Clones produced as described in Example 14 were grown to mid-log phase,induced with 1 mM IPTG and grown for four hours. Cells were pelleted,washed, and lysed in boiling SDS-PAGE loading buffer. Crude lysates wereseparated by SDS-PAGE and Coomassie stained. A second set of separatedproteins was transferred to PVDF membrane and immunblotted with IRPEvaccine hyperimmunized mouse sera diluted 1:500 in 1% NFDM/TTBS. Theblot was washed, incubated with alkaline phosphatase (AP)-conjugatedgoat anti-mouse secondary antibody, washed, and developed withchromogenic substrate.

Example 16 Preparation of Immunizing Compositions fromRecombinantly-Produced Polypeptides

In order to isolate recombinant S. aureus polypeptides for formulating avaccine, the E. coli clones described in Example 14 were grown at 37° C.(225 rpm) to mid-log phase (OD₆₀₀=0.4-0.6) in 1 L of Trypic Soy Brothand then induced for 4 hours with 1 mM isopropylβ-D-1-thiogalactopyranoside (IPTG). Cultures were pelleted for 10minutes at 4° C. in a Sorvall centrifuge (4000×g) and frozen at −80° C.prior to undergoing the purification procedure. Bacterial pellets werethen processed by two different methods, depending on the solubility ofthe over-expressed S. aureus polypeptide.

For soluble polypeptides (e.g., MntC, FhuD, SYN2, SirA, or SYN1),bacterial pellets were resuspended in 25 ml of BUGBUSTER reagent(Novagen) and subjected to 15 minutes of sonication on ice using aBranson sonifier fitted with a microtip (65% duty cycle, 5 output).Insoluble material was removed by 10 minutes of centrifugation (4000×g).The soluble supernatant was filtered (0.2 μm) and subjected to metalaffinity chromatography (Ni-NTA His-Bind, Novagen) according to theinstructions provided by the manufacturer.

For insoluble polypeptides (e.g., PflB, or Opp1A), bacterial pelletswere resuspended in 25 ml of BUGBUSTER reagent (Novagen), placed on arocker platform for 10 minutes, and then subjected to centrifugation(15,000×g for 12 minutes). The resulting pellet was resuspended in 10 mlof BUGBUSTER plus 20 ml of diluted BUGBUSTER (1:10 in PBS) and subjectedto centrifugation (5000×g for 12 minutes). The pellet was thenresuspended in 20 ml of diluted BUGBUSTER and subjected to a final stepof centrifugation (15,000×g for 12 minutes). The final pellet wasresuspended in 10 ml of Buffer A (0.1 M NaH₂PO₄, 0.01 Tris-HCl, 8M Urea,pH 8.0) and incubated for 10 minutes on a rocking platform at roomtemperature. The samples were then subjected to centrifugation (12,000×gfor 20 minutes), and the resulting supernatant was separated by metalaffinity chromatography (Ni-NTA His-Bind, Novagen) according theinstructions provided by the manufacturer, but with the followingmodifications. After charging the column, 10 ml of Buffer A was used toequilibrate the resin. Following the binding of polypeptide, the columnwas washed with 15 ml of Buffer B (0.1 M NaH₂PO₄, 0.01 Tris-HCl, 8MUrea, pH 6.0), and eluted using 15 ml of Buffer C (0.1 M NaH₂PO₄, 0.01Tris-HCl, 8M Urea, pH 4.5).

Isolated recombinant polypeptides were eluted from the columns in avolume of 15 ml and placed in 20 kDa cutoff dialysis cassettes (Pierce)for dialysis against 2 L of phosphate-buffered saline (PBS). Followingthree buffer changes over the course of 30 hours, the polypeptides wereremoved, filtered (0.2 μm), and concentrated to 2-3 ml of volume using20 kDa cutoff Centricon devices (Millipore). The concentrations of thepurified polypeptides were determined using the standard BCA method(Pierce).

10 μg of each polypeptide was combined, and the volume adjusted to 100μl with PBS, to form an immunizing composition.

Example 17 Mouse Vaccination IV Challenge (Study A)

Fifty (N=50) female BALB/C mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams were distributedinto 3 groups (10-20 mice/group). Mice were housed in polycarbonatemouse cages (N=5 mice per cage). Food and water were supplied ad libitumto all mice. All vaccines were formulated with 50% IFA as an adjuvant.Mice were vaccinated subcutaneously with 0.1 ml of the appropriatecomposition two times at 14 day intervals as follows:

Group 1: Placebo, vaccinated with ovalbumin (70 μg/100 μl) (Placebo, 20mice).

Group 2: Vaccinated with ATCC 25904 proteins expressed underiron-restriction (70 μg/100 μl) (SIRP Extract, 20 mice).

Group 3: Vaccinated with recombinant polypeptide MntC (10 μg/100 μl)(rMntC, 10 mice).

IP Challenge (Study B)

Forty (N=40) female BALB/C mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams were equallydistributed into 4 groups (10 mice/group). Mice were housed inpolycarbonate mouse cages (N=5 mice per cage). Food and water weresupplied ad libitum to all mice. All vaccines were formulated with 50%IFA as an adjuvant. Mice were vaccinated subcutaneously with 0.1 ml ofthe appropriate composition at 14 day intervals using either twovaccinations (Groups 1-3) or three vaccinations (Group 4) as follows:

Group 1: Placebo, vaccinated twice with ovalbumin (70 μg/100 μl)(Placebo).

Group 2: Vaccinated twice with ATCC 25904 proteins expressed underiron-restriction (70 μg/100 μl) (SIRP Extract).

Group 3: Vaccinated twice with recombinant polypeptides PflB, Opp1A,SirA, SYN2, FhuD, SYN1, and MntC (each 10 μg/100 μl, total protein 70μg/100 μl) (rSIRP7 (2×)).

Group 4: Vaccinated three times with recombinant polypeptides PflB,Opp1A, SirA, SYN2, FhuD, SYN1, and MntC (each 10 μg/100 μl, totalprotein 70 μg/100 (rSIRP7 (3×)).

IC Challenge (Study C)

Thirty (N=30) female BALB/C mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams were equallydistributed into 3 groups (10 mice/group). Mice were housed inpolycarbonate mouse cages (N=5 mice per cage). Food and water weresupplied ad libitum to all mice. Mice were vaccinated subcutaneouslywith 0.1 ml of the appropriate composition two times at 14 day intervalsas follows:

Group 1: Placebo, vaccinated with ovalbumin (70m/100 μl) (Placebo).

Group 2: Vaccinated with ATCC 25904 proteins expressed underiron-restriction (70 μg/100 μl) (SIRP Extract).

Group 3: Vaccinated with recombinant polypeptides PflB, Opp1A, SirA,SYN2, FhuD, SYN1, and MntC (each 10 μg/100 μl, total protein 70 μg/100μl) (rSIRP7).

Example 18 Preparation of Challenge Organism IV Challenge (Study A)

Staphylococcus aureus strain ATCC 25904 was used as a challengeorganism. Briefly, a 1 μl loop of bacteria from a frozen glycerol stockgrown in standard TSB (no iron restriction) was used to inoculate a 20ml culture of TSB and incubated at 37° C. for 18 hours. 2.5 ml of thisculture were passaged into 500 ml of fresh TSB. The culture wasincubated at 37° C. for approximately two hours while rotating at 250rpm until an optical density (OD₆₀₀) of 0.4 (Absorbance) was reached(mid-log phase), then the cells were centrifuged at 10,000×g for 10minutes at 4° C. to pellet the bacteria. The bacterial pellet was washedby centrifugation in PBS at 4° C. The final pellet was resuspended in 20ml of PBS. The final challenge dose was prepared by adding an aliquot ofthis concentrated bacterial culture to PBS to generate a solution withan OD₆₀₀ of 4.0 (A), corresponding to approximately 6.67×10⁸ CFU/ml.Just prior to challenge, 1 ml of these bacterial suspensions wasserially diluted and plated on agar to enumerate the number ofcolony-forming units (CFU) per mouse dose.

IP Challenge (Study B)

Staphylococcus aureus strain ATCC 25904 was used as a challengeorganism. Briefly, a 1 μl loop of bacteria from a frozen glycerol stockgrown in standard TSB (no iron restriction) was used to inoculate a 20ml culture of TSB and incubated at 37° C. for 18 hours. 2.5 ml of thisculture were passaged into 500 ml of fresh TSB. The culture wasincubated at 37° C. for approximately two hours while rotating at 250rpm until an optical density (OD₆₀₀) of 0.4 (Absorbance) was reached(mid-log phase), then the cells were centrifuged at 10,000×g for 10minutes at 4° C. to pellet the bacteria. The bacterial pellet was washedby centrifugation in PBS at 4° C. The final pellet was resuspended in 20ml of PBS. The final challenge dose was prepared by adding an aliquot ofthis concentrated bacterial culture to PBS to generate a solution withan OD₆₀₀ of 6.0 (A), corresponding to approximately 3.33×10⁹ CFU/ml.Just prior to challenge, 1 ml of these bacterial suspensions wasserially diluted and plated on agar to enumerate the number ofcolony-forming units (CFU) per mouse dose.

IV Challenge (Study C)

Staphylococcus aureus strain ATCC 25904 was used as a challengeorganism. Briefly, a 1 μl loop of bacteria from a frozen glycerol stockgrown in standard TSB (no iron restriction) was used to inoculate a 20ml culture of TSB and incubated at 37° C. for 18 hours. 2.5 ml of thisculture were passaged into 500 ml of fresh TSB. The culture wasincubated at 37° C. for approximately two hours while rotating at 250rpm until an optical density (OD₆₀₀) of 0.4 (Absorbance) was reached(mid-log phase), then the cells were centrifuged at 10,000×g for 10minutes at 4° C. to pellet the bacteria. The bacterial pellet was washedby centrifugation in PBS at 4° C. The final pellet was resuspended in 20ml of PBS. The final challenge dose was prepared by adding an aliquot ofthis concentrated bacterial culture to PBS to generate a solution withan OD₆₀₀ of 4.0 (A), corresponding to approximately 6.67×10⁸ CFU/ml.Just prior to challenge, 1 ml of these bacterial suspensions wasserially diluted and plated on agar to enumerate the number ofcolony-forming units (CFU) per mouse dose.

Example 19 Challenge IV Challenge (Study A)

Fourteen days after the second vaccination, mice in all groups (1-3)were intravenously challenged in the lateral tail vein with 0.3 ml ofthe appropriate organism. The three groups of mice were challengedidentically with 2×10⁸ CFU of S. aureus strain ATCC 25904 per mouse.Mortality was recorded daily for 10 days after challenge.

When comparing the mice challenged with the ATCC 25904 isolate, 80% ofthe placebo-vaccinated Group 1 mice died within 10 days of challenge(Table 14). This demonstrated that strain ATCC 25904 caused a high rateof mortality in mice at the dose level administered. In contrast to themice in Group 1, only 25% of the mice in Group 2 (vaccinated withproteins extracted from strain ATCC 25904 following growth iniron-depleted conditions, SIRP Extract) died within 10 dayspost-challenge. These results illustrated that the mice challenged withstrain ATCC 25904 were significantly protected by vaccination with theprotein composition derived from iron-depleted ATCC 25904 (p=0.0006,logrank test for mortality). In addition, only 50% of the mice in Group3 (vaccinated with recombinant MntC polypeptides, rMntC) died within 10days of challenge, indicating that recombinant proteins offeredprotection against challenge with the ATCC 25904 strain (p=0.100,logrank test for mortality).

IP Challenge (Study B)

Fourteen days after the second vaccination, mice in all groups (1-4)were intraperitoneally challenged with 0.5 ml of the appropriateorganism. The three groups of mice were challenged identically with1×10⁹ CFU of S. aureus strain ATCC 25904 per mouse. Mortality wasrecorded daily for 10 days after challenge.

When comparing the mice challenged with the ATCC 25904 isolate, 60% ofthe placebo-vaccinated Group 1 mice died within 10 days of challenge(Table 14). This demonstrated that strain ATCC 25904 caused a moderaterate of mortality in mice at the dose level administered. In contrast tothe mice in Group 1, only 30% of the mice in Group 2 (vaccinated withproteins extracted from strain ATCC 25904 following growth iniron-depleted conditions, SIRP Extract) died within 10 dayspost-challenge. These results illustrated that the mice vaccinated withthe protein composition derived from iron-depleted ATCC 25904 died athalf the rate of placebo-vaccinated mice when challenged with strainATCC 25904 (p=0.143, logrank test for mortality). Mice vaccinated withthe combination of seven recombinant proteins showed a higher level ofprotection relative to placebo. Only 20% of the mice in Group 3(vaccinated 2× with recombinant polypeptides, rSIRP7 2×) died within 10days of challenge and only 10% of the mice in Group 4 (vaccinated 3×with recombinant polypeptides, rSIRP7 3×) died within 10 days ofchallenge, indicating that three vaccinations with recombinant proteinsoffered significant protection against challenge with the ATCC 25904strain (p=0.040, logrank test for mortality).

IV Challenge (Study C)

Fourteen days after the second vaccination, mice in all groups (1-3)were intravenously challenged in the lateral tail vein with 0.3 ml ofthe appropriate organism. The three groups of mice were challengedidentically with 2×10⁸ CFU of S. aureus strain ATCC 25904. Mortality wasrecorded daily for 10 days after challenge.

When comparing the mice challenged with the ATCC 25904 isolate, 90% ofthe placebo-vaccinated Group 1 mice died within 10 days of challenge(Table 14). This demonstrated that strain ATCC 25904 caused a high rateof mortality in mice at the dose level administered. In contrast to themice in Group 1, only 40% of the mice in Group 2 (vaccinated withproteins extracted from strain ATCC 25904 following growth iniron-depleted conditions, SIRP Extract) died within 10 dayspost-challenge. These results illustrated that the mice challenged withstrain ATCC 25904 were significantly protected by vaccination with theprotein composition derived from iron-depleted ATCC 25904 (p=0.0164,logrank test for mortality). In addition, only 40% of the mice in Group3 (vaccinated with recombinant polypeptides, rSIRP7) died within 10 daysof challenge, indicating that recombinant proteins offered significantprotection against challenge with the ATCC 25904 strain (p=0.0255,logrank test for mortality).

Results are shown in Table 14 and FIG. 161.

TABLE 14 Mortality of Vaccinated and Non-Vaccinated Mice FollowingChallenge with Staphylococcus aureus ATCC isolate 25904. Groups # Mice #Dead Percent mortality (%) IV Challenge (Study A) Group 1 (Placebo) 2016/20  80 Group 2 (SIRP Extract) 10 5/20 25 Group 3 (rMntC) 10 5/10 50IP Challenge (Study B) Group 1 (Placebo) 10 6/10 60 Group 2 (SIRPExtract) 10 3/10 30 Group 3 (rSIRP (2x)) 10 2/10 20 Group 4 (rSIRP (3x))10 1/10 10 IV Challenge (Study C) Group 1 (Placebo) 10 9/10 90 Group 2(SIRP Extract) 10 4/10 40 Group 3 (rMntC) 10 4/10 40

Example 20 Passive Immunization Using Recombinantly-PreparedPolypeptides

A polyclonal antibody composition is prepared as described in Example 8,except that the mice are vaccinated with a composition of recombinantpolypeptides prepared as described in Example 16.

The resulting antibody composition is used to passively immunize mice asdescribed in Example 9. The immunized mice are challenged as describedin Example 9.

Immunized mice will show decreased mortality compared to unvaccinatedand placebo vaccinated mice.

Example 21 Large Scale Fermentation and Isolation ofRecombinantly-Produced Polypeptides

A master seed stock of the recombinant E. coli of Example 14 can beprepared by growing the organism in 2000 ml of sterile RM medium (20 gcasamino acids, 60 g Na₂HPO₄, 30 g KH₂PO₄, 5 g NaCl 10 g NH₄Cl per literand 100 ug/ml ampicillin) for 8 hours at 37° C. The bacteria can beharvested by centrifugation at 10,000×g for 30 minutes. The culture canbe washed twice by centrifugation (10,000×g) and the final bacterialpellet resuspended in 500 ml of sterile RM medium containing 20% sterileglycerol. One milliliter of the culture will be transferred to a 2 mlcryovial and stored at −85° C.

A cryovial (1 ml) of the recombinant master seed stock can be used toinoculate a 100 ml culture flask containing the medium described above,with the exception of having 2 g casamino acids and 0.5% glucose(“modified RM medium”). The culture can be incubated at 37° C. for sevenhours, at which time it can be inoculated into 2 liters of modified RMmedium and allowed to grow for an additional four hours at 37° C. Thisculture can be used to inoculate a 30-liter New Brunswick BIOFLOW 4bench-top fermenter charged with 20 liters of modified RM medium exceptthe final concentration of casamino acids will be 20 g/liter(“fermentation RM medium”). The pH of the fermentation medium can beheld maintained between 6.9 and 7.2 by automatic titration with 30% NaOHand 10% HCl. The fermentation culture can be stirred at 350 rev/minute,and the culture can be aerated with 11 liters/minute at 37° C. Foamingcan be controlled automatically by the addition of 0.4% siliconedefoamer (Antifoam-B, J. T. Baker, N.J.). The culture can be allowed togrow continuously at these conditions for four hours (OD₆₀₀=4.0-6.0), atwhich time the culture can be pumped into a 150-liter fermenter (W. B.Moore, Easton PN), charged with 110 liters of fermentation RM medium and0.2% defoamer. The parameters of the fermentation can be as follows: 650rpm, 60% DO, 50 slpm air, 10 psi back pressure, 37° C., and pH held at7.2 with NaOH. After reaching late exponential phase growth(approximately six hours, OD₆₀₀=15.0), recombinant proteins can beinduced by adding of 150 ug/ml IPTG. The fermentation can be allowed togrow for an additional three hours at which point the fermentation canbe terminated by lowing the temperature of the fermentor to 18° C.(OD₆₀₀ 20-25 at a 1:100 dilution).

After fermentation, the recombinantly-produced polypeptides can beharvested by conventional means. The cells are disrupted (e.g., byhomogenization) to release the recombinantly-produced polypeptides, someof which are soluble and some of which are insoluble.

Soluble polypeptides can be concentrated by tangential flow filtration,detergent solubilized, and harvested by metal affinity chromatography.The collected soluble polypeptides can be isolated as described for theisolation of soluble polypeptides in Example 16.

Insoluble proteins can be harvested by high speed centrifugation. Thepellet of insoluble polypeptides can be collected and then isolated asdescribed for the isolation of insoluble polypeptides in Example 16.

Example 22 Mouse Vaccination

Recombinantly-produced SirA, SYN2, FhuD, and MntC polypeptides areprepared and isolated as described in Example 16.

Thirty (N=30) female BALB/C mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams are equallydistributed into three groups (10 mice/group). Mice are housed inpolycarbonate mouse cages (N=5 mice per cage). Food and water aresupplied ad libitum to all mice. Mice are vaccinated subcutaneously with0.1 ml of the appropriate composition two times at 14 day intervals asfollows:

Group-1: Placebo, vaccinated with ovalbumin (40 μm/100 μl)

Group-2: Vaccinated with ATCC 25904 proteins expressed underiron-restriction (40 μg/100 μl).

Group-3: Vaccinated with recombinant polypeptides SirA, SYN2, FhuD, andMntC (each 10 μg/100 μl, total protein 40 μg/100 μl).

Mice are challenged as described in Example 19. Mice in Group 3 willexhibit reduced mortality compared to mice in Group 1.

Example 23 Mouse Vaccination

Recombinantly-produced PflB polypeptide is prepared and isolated asdescribed in Example 16.

Thirty (N=30) female BALB/C mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams are equallydistributed into three groups (10 mice/group). Mice are housed inpolycarbonate mouse cages (N=5 mice per cage). Food and water aresupplied ad libitum to all mice. Mice are vaccinated subcutaneously with0.1 ml of the appropriate composition two times at 14 day intervals asfollows:

Group-1: Placebo, vaccinated with ovalbumin (10 μg/100 μl)

Group-2: Vaccinated with ATCC 25904 proteins expressed underiron-restriction (10 μg/100

Group-3: Vaccinated with recombinant polypeptide PflB (each 10 μg/100μl).

Mice are challenged as described in Example 19. Mice in Group 3 willexhibit reduced mortality compared to mice in Group 1.

Example 24 Mouse Vaccination

Recombinantly-produced Opp1A polypeptide is prepared and isolated asdescribed in Example 16.

Thirty (N=30) female BALB/C mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams are equallydistributed into three groups (10 mice/group). Mice are housed inpolycarbonate mouse cages (N=5 mice per cage). Food and water aresupplied ad libitum to all mice. Mice are vaccinated subcutaneously with0.1 ml of the appropriate composition two times at 14 day intervals asfollows:

Group-1: Placebo, vaccinated with ovalbumin (10 μg/100 μl)

Group-2: Vaccinated with ATCC 25904 proteins expressed underiron-restriction (10 μg/100 μl).

Group-3: Vaccinated with recombinant polypeptide Opp1A (each 10 μg/100μl).

Mice are challenged as described in Example 19. Mice in Group 3 willexhibit reduced mortality compared to mice in Group 1.

Example 25 Mouse Vaccination

Recombinantly-produced SirA polypeptide is prepared and isolated asdescribed in Example 16.

Thirty (N=30) female BALB/C mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams are equallydistributed into three groups (10 mice/group). Mice are housed inpolycarbonate mouse cages (N=5 mice per cage). Food and water aresupplied ad libitum to all mice. Mice are vaccinated subcutaneously with0.1 ml of the appropriate composition two times at 14 day intervals asfollows:

Group-1: Placebo, vaccinated with ovalbumin (10 μg/100 μl)

Group-2: Vaccinated with ATCC 25904 proteins expressed underiron-restriction (10 μg/100 μl).

Group-3: Vaccinated with recombinant polypeptide SirA (each 10 μg/100μl).

Mice are challenged as described in Example 19. Mice in Group 3 willexhibit reduced mortality compared to mice in Group 1.

Example 26 Mouse Vaccination

Recombinantly-produced SYN2 polypeptide is prepared and isolated asdescribed in Example 16.

Thirty (N=30) female BALB/C mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams are equallydistributed into three groups (10 mice/group). Mice are housed inpolycarbonate mouse cages (N=5 mice per cage). Food and water aresupplied ad libitum to all mice. Mice are vaccinated subcutaneously with0.1 ml of the appropriate composition two times at 14 day intervals asfollows:

Group-1: Placebo, vaccinated with ovalbumin (10 μg/100 μl)

Group-2: Vaccinated with ATCC 25904 proteins expressed underiron-restriction (10 μg/100 μl).

Group-3: Vaccinated with recombinant polypeptide SYN2 (each 10 μg/100μl).

Mice are challenged as described in Example 19. Mice in Group 3 willexhibit reduced mortality compared to mice in Group 1.

Example 27 Mouse Vaccination

Recombinantly-produced FhuD polypeptide is prepared and isolated asdescribed in Example 16.

Thirty (N=30) female BALB/C mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams are equallydistributed into three groups (10 mice/group). Mice are housed inpolycarbonate mouse cages (N=5 mice per cage). Food and water aresupplied ad libitum to all mice. Mice are vaccinated subcutaneously with0.1 ml of the appropriate composition two times at 14 day intervals asfollows:

Group-1: Placebo, vaccinated with ovalbumin (10 μg/100 μl)

Group-2: Vaccinated with ATCC 25904 proteins expressed underiron-restriction (10 μg/100 μl).

Group-3: Vaccinated with recombinant polypeptide FhuD (each 10 μg/100μl).

Mice are challenged as described in Example 19. Mice in Group 3 willexhibit reduced mortality compared to mice in Group 1.

Example 28 Mouse Vaccination

Recombinantly-produced SYN1 polypeptide is prepared and isolated asdescribed in Example 16.

Thirty (N=30) female BALB/C mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams are equallydistributed into three groups (10 mice/group). Mice are housed inpolycarbonate mouse cages (N=5 mice per cage). Food and water aresupplied ad libitum to all mice. Mice are vaccinated subcutaneously with0.1 ml of the appropriate composition two times at 14 day intervals asfollows:

Group-1: Placebo, vaccinated with ovalbumin (10 μg/100 μl)

Group-2: Vaccinated with ATCC 25904 proteins expressed underiron-restriction (10 μg/100

Group-3: Vaccinated with recombinant polypeptide SYN1 (each 10 μg/100μl).

Mice are challenged as described in Example 19. Mice in Group 3 willexhibit reduced mortality compared to mice in Group 1.

Example 29 Mouse Vaccination

Recombinantly-produced MntC polypeptide is prepared and isolated asdescribed in Example 16.

Thirty (N=30) female BALB/C mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams are equallydistributed into three groups (10 mice/group). Mice are housed inpolycarbonate mouse cages (N=5 mice per cage). Food and water aresupplied ad libitum to all mice. Mice are vaccinated subcutaneously with0.1 ml of the appropriate composition two times at 14 day intervals asfollows:

Group-1: Placebo, vaccinated with ovalbumin (10 μg/100 μl)

Group-2: Vaccinated with ATCC 25904 proteins expressed underiron-restriction (10 μg/100 μl).

Group-3: Vaccinated with recombinant polypeptide MntC (each 10 μg/100μl).

Mice are challenged as described in Example 19. Mice in Group 3 willexhibit reduced mortality compared to mice in Group 1.

Example 30 Mouse Vaccination

Recombinantly-produced SstD polypeptide is prepared and isolated asdescribed in Example 16.

Thirty (N=30) female BALB/C mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams are equallydistributed into three groups (10 mice/group). Mice are housed inpolycarbonate mouse cages (N=5 mice per cage). Food and water aresupplied ad libitum to all mice. Mice are vaccinated subcutaneously with0.1 ml of the appropriate composition two times at 14 day intervals asfollows:

Group-1: Placebo, vaccinated with ovalbumin (10 μg/100 μl)

Group-2: Vaccinated with ATCC 25904 proteins expressed underiron-restriction (10 μg/100 μl).

Group-3: Vaccinated with recombinant polypeptide SstD (each 10 μg/100μl).

Mice are challenged as described in Example 19. Mice in Group 3 willexhibit reduced mortality compared to mice in Group 1.

Example 31 Mouse Vaccination

Recombinantly-produced FhuD2 polypeptide is prepared and isolated asdescribed in Example 16.

Thirty (N=30) female BALB/C mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams are equallydistributed into three groups (10 mice/group). Mice are housed inpolycarbonate mouse cages (N=5 mice per cage). Food and water aresupplied ad libitum to all mice. Mice are vaccinated subcutaneously with0.1 ml of the appropriate composition two times at 14 day intervals asfollows:

Group-1: Placebo, vaccinated with ovalbumin (10 μg/100 μl)

Group-2: Vaccinated with ATCC 25904 proteins expressed underiron-restriction (10 μg/100

Group-3: Vaccinated with recombinant polypeptide FhuD2 (each 10 μg/100μl).

Mice are challenged as described in Example 19. Mice in Group 3 willexhibit reduced mortality compared to mice in Group 1.

Example 32 Recombinant Polypeptides are Specifically Bound by SeraGenerated by S. aureus Infection

S. aureus were prepared as challenge organisms as described in Example 4and used to challenge mice as described in Examples 5 or 19. Sera wereobtained from mice either prior to challenge or from mice which hadreceived placebo vaccine, had been challenged with S. aureus Newman andrecovered (convalescent). Blood was collected from challenged andunchallenged mice and sera were obtained by centrifugation. Sera wereused to evaluate the reactivity of the individual recombinantly-producedpolypeptides using an immunoblot method as described in Example 15.

Sera collected from mice prior to challenge did not react with any ofthe recombinant polypeptides.

Antibody in the sera produced as a result of S. aureus challenge reactedwith the recombinantly-produced polypeptides as shown in FIG. 202.Antibody raised against polypeptides expressed during infection by S.aureus recognizes recombinantly-produced vairiants of the polypeptides.Thus, recombinant polypeptides are immunological substitutes for—e.g.,immunological fragments of—immunological polypeptides expressed by thechallenge S. aureus.

Example 33 Antibody Against Recombinantly-Produced Polypeptides andIron-Regulated Membrane Polypeptides Extracted Directly from S. aureusCells Crossreact

Recombinant iron-regulated polypeptides are expressed and purified asdescribed in Example 16. Iron-regulated membrane polypeptides areextracted directly from S. aureus cells grown in low-iron conditions asdescribed in Example 1. The recombinant polypeptides are formulated intoa vaccine as described in Example 16 and used to vaccinate mice asdescribed in Example 17. The S. aureus-extracted polypeptides areformulated into vaccines as described in Example 2 and used to vaccinatemice as described in Example 3. For the recombinant polypeptidevaccines, a single recombinant polypeptide will be formulated into thevaccine. Antisera from vaccinated mice are collected and used forimmunoblots as described in Example 15.

The antisera from animals vaccinated with recombinantly-producediron-regulated polypeptides are further used to evaluate reactivity withextracted iron-regulated polypeptides following the separation of theextracted iron-regulated polypeptides by SDS-PAGE. Antisera from animalsvaccinated with recombinant polypeptides will bind to appropriateSDS-PAGE-separated extracted polypeptides where antibody epitopes areconserved between the extracted polypeptides and therecombinantly-produced polypeptides.

The antisera from animals vaccinated with extracted iron-regulatedpolypeptides are further used to evaluate reactivity with individualrecombinantly-produced iron-regulated polypeptides following theseparation of the recombinant iron-regulated polypeptides by SDS-PAGE.Antisera from animals vaccinated with extracted polypeptides will bindto appropriate SDS-PAGE-separated recombinant polypeptides whereantibody epitopes are conserved between the recombinantly-producedpolypeptides and the extracted polypeptides.

Example 34 Western Blot Analysis of Recombinant S. aureus Proteins

Recombinant S. aureus proteins were prepared as described in Example 16,then subjected to sodium dodecyl sulfate-10% polyacrylamide gelelectrophoresis and transferred to nitrocellulose membranes (BioRad,Hercules, Calif.) for Western blot analysis. Individual blots werereacted with serum from healthy (no reported Staphylococcus infection)or convalescent (Methicilin Resistant S. aureus) human donors.

As a control to identify each recombinant Histidine-tagged protein, eachblot was co-incubated with anti-Histidine antibodies (RocklandImmunochemicals, Inc., Gilbertsville, Pa. and Qiagen GmbH, Hilden,Germany) to identify the recombinant proteins using two-color analysis.All primary antisera were used at a 1:1000 dilution and incubated for 1hour on a rocker. Following several washes with TBS+0.05% Tween toremove unbound antibody, the membranes were subsequently incubated witha 800CW dye-conjugated human secondary antibody (RocklandImmunochemicals, Inc., Gilbertsville, Pa.), 680 dye-conjugated mousesecondary antibody (Li-cor Biosciences, Lincoln, Nebr.) or a 800CWdye-conjugated rabbit secondary antibody (Li-Cor Biosciences) at thedilution recommended by the manufacturer, for 1 hour, in the dark. Themembranes were washed an additional three to five times with TBS+0.05%Tween with the last wash in TBS-only. Fluorescent signals (680 and 800)were detected using the Odyssey Infrared Imaging System (Li-CorBiosciences). Results are shown In FIG. 203 (healthy) and FIG. 204(convalescent).

Example 35 S. aureus DU5875 Surface Expression of Metal-RegualtedPolypeptides

S. aureus strain DU5875 (cap-, spa-) was grown in iron-replete (TSB+0.3mM ferric chloride) or iron-deplete (TSB+1 mM dipyridyl) conditions toan OD₆₀₀ of approximately 0.6.

In panels A-C of FIG. 205, S. aureus strain DU5875 was grown iniron-deplete conditions to an OD₆₀₀ of approximately 0.75 and frozen.Bacteria were thawed, washed in PBS and resuspended in PBS+1% Pig IgG+1%BSA as a blocking step. Mouse antiserum raised against FhuD, Opp1A orPflB was used at a dilution of 1:100 to stain approximately 2×10⁶bacteria. Preimmune mouse serum was used as a negative control. Bacteriawere then washed in blocking buffer and incubated with anAF633-conjugated goat anti-mouse secondary antibody, and analyzed on aflow cytometer. Bacteria incubated with preimmune mouse serum, at a1:100, were used as a negative control.

In panel D of FIG. 205, S. aureus strain DU5875 was grown iniron-deplete conditions to an OD₆₀₀ of approximately 2.0 and frozen.Bacteria were thawed, washed in PBS and resuspended in PBS+0.2% PigIgG+1% BSA as a blocking step. Mouse antiserum raised against rSIRP7 wasused at a dilution of 1:50 to stain approximately 5×10⁷ bacteria, withpreimmune mouse serum used as a negative control. Bacteria were thenwashed in blocking buffer and incubated with an AF633-conjugated goatanti-mouse secondary antibody, and analyzed on a flow cytometer.

The results of this assay indicate that murine antibodies raised againstthe SIRP proteins bind to S. aureus cells. Cells grown underiron-deplete conditions bind more antibody than cells grown underiron-replete conditions, providing further evidence that FhuD, Opp1A,and PflB are expressed at higher levels under low-iron conditions andare antigens that can induce immunological activity againstStaphylococcus spp. The increase in median fluorescence intensity (MFI)demonstrates the relative increase in fluorescence when anti-SIRPantibodies bind to S. aureus cells compared to the MFI of preimmunemouse serum. Results are shown in FIG. 205.

Example 36

A Luminex assay was used to evaluate cytokine expression by splenocytesfrom mice immunized with the combination (rSIRP7) of recombinant SIRPcomponents PflB (SEQ ID NO:353), Opp1A (SEQ ID NO:364), SirA (SEQ IDNO:375), SYN2 (SEQ ID NO:386), FhuD (SEQ ID NO:397), SYN1 (SEQ IDNO:408, and MntC (SEQ ID NO:419) or placebo, then restimulated with SIRPEextract (SIRPE) or rSIRP7. Several cytokines were upregulated uponrestimulation, and the cytokine profiles induced by SIRPE and rSIRP7restimulation were similar. The overall cytokine profile in response torSIRP7 or SIRPE restimulation resembled that expected from aTh1/Th17-type immune response, and demonstrates that vaccination withthe recombinant SIRP components induces a cellular immune response thatcan be measured based on cytokine expression.

Methods. Mice were vaccinated two times, 14 days apart, with 70 totalprotein (OVA, SIRP Extract, or rSIRP7) formulated with 50% IFA. CD4⁺ Tcells were purified from splenocyte suspensions by negative selectionusing a CD4⁺ T cell isolation kit and LD columns (Miltenyi Biotec, Inc.,Auburn, Calif.). Briefly, biotinylated antibodies were used to label allcells except CD4⁺ T cells, and then streptavidin-conjugated magneticbeads were used to remove these cells from the mixture with a magneticcolumn, leaving highly purified CD4⁺ T cells. The resulting CD4⁺ T cellswere found to be greater than 95% pure based on CD3 and CD4 expression.Naïve splenocytes were treated with Mitomycin-C to generate mitoticallyinactive antigen presenting cells. 4×10⁵ APC added to 5×10⁵CD4⁺ T cellsplus stimulation antigen, followed by 42 hours of incubation.Supernatants were analyzed by Luminex using the standard assayparameters. Results are shown in FIG. 206.

Example 37

S. aureus extracts from several strains (Newman, Reynolds) were preparedusing the method described in Example 1. S. aureus cells were grown ineither iron restricted media containing 1000 μm 2,2-dipyridyl(Sigma-Aldrich St. Louis, Mo.) or iron replete media containing 300 μMFeCl3 (Sigma-Aldrich St. Louis, Mo.).

Proteins within S. aureus membrane extracts from iron-replete andiron-deplete cultures were identified and quantified using ITRAQ and LCQmass spectrometry. Amine-modified labeling of membrane extracts for irondeficient and iron replete Staphylococcus aureus Newman strain wereperformed with ITRAQ-8plex reagents (Applied Biosystems, Inc., FosterCity, Calif.) using 40.0 μg of membrane extract (reagents 113 versus115) according to the manufacturer's 8Plex protocol. Cation exchangechromatography was applied using an MCX column (Waters Corp., Milford,Mass.) and the peptides separated using an ULTIMATE 3000 NANO LC system(Dionex Corp. Bannockburn, Ill.) coupled to ESI mode using a QSTAR XLmass spectrometer (Applied Biosystems, Inc., Foster City, Calif.).

The ratio of metal-regulated polypeptides produced by cells grown iniron-restricted media compared to iron-replete media was measured. Theratio is a relative measure of protein expression and does not providedata indicating an absolute amount of protein present in the extract.Results are shown in Table 15.

TABLE 15 Protein Identified in extract Fold increase in low iron MntCYes 22 SYN1 Yes Not determined FhuD No Not determined SYN2 Yes 23 SirAYes 36 Opp1A Yes Not determined PflB Yes Not determined FhuD2 Yes  6SstD Yes 14

Example 38

An oxidative burst assay can be used to measure the production ofreactive oxygen species by neutrophils, an indication of an inflammatoryresponse. To obtain neutrophils from fresh blood, red blood cells fromfresh human blood are lysed by the addition of lysis buffer (150 mMNH₄Cl, 10 mM KHCO₃, 1 mM disodium EDTA, pH 7.4) at a 1:10 dilution,incubated for 10 minutes at room temperature and centrifuged for 10minutes at 430×g. The supernatant is removed by tube inversion andpellets are washed twice with PBS, then resuspended in 5 ml ofRPMI-Hepes 5% FCS+Glutamine (complete RPMI) and enumerated using aMULTISIZER (Beckman Coulter, Inc., Brea, Calif.) after a 1/500 dilutionof cell suspension in ISOTON (20 μl of cells in 10 ml ISOTON, BeckmanCoulter, Inc., Brea, Calif.).

For the preparation of bacteria, S. aureus strain Lowenstein is seededin TSB medium and grown for 20 hours at 37° C. in 50 ml of medium. Fromthis culture, 5 ml are pelleted for 10 minutes at 4000 rpm at 4° C. Thepellet is then washed with 50 ml of PBS and repelleted by centrifugationfor 10 minutes at 4000 rpm at 4° C. The wash step is repeated and thebacterial pellet is resuspended in 5 ml of PBS. Bacteria are adjusted toa theoretical density of 1.10⁹ CFU/ml and cell dilutions are plated onagar and incubated for exact enumeration the following day.

To decomplement the sera, all sera are incubated for 30 minutes at 56°C. Mixtures of sera and cells are performed in polypropylene sterile DWplates in a final volume of 500 μl per well. Into each well, thefollowing reagents are added (as shown in Table 16), in order: culturemedium (RPMI-Hepes, glutamine, 5% FCS), live bacteria at the appropriateconcentration, the sera at the appropriate dilution, the complement,hPMNs at the appropriate concentration and, finally, the DHR molecule(Life Technologies, Inc., Carlsbad, Calif.) as the marker of oxidativeburst. Plates are incubated for 25 minutes at 37° C. with shaking. Thereaction is stopped by incubating the plates for five minutes on ice.

TABLE 16 Final Working Vol./ concentration Reagent Identification Conc.well (in 500 μl) Live bacteria TSB, 20 h, 37° C. 1.25 · 10⁸ 200 μl  5 ·10⁷ 10⁹ CFU/ml CFU/ml CFU/ml Whole blood From 2 different 2.5 · 10⁶ 100μl  0.5 · 10⁶ leukocytes donors after the cells/ml cells/ml red bloodcell lysis DHR Life Technologies, 100 50 μl 10 Inc. Cat. No. D632 μg/mlμg/ml (10 mg/ml) Baby rabbit Produced in-house 1/10 50 μl 1/100 (1%)complement Sera Serum from mouse 1/10 50 μl 1/100  immunized withadjuvant alone Anti-whole cell  1/100 50 μl 1/1000 control Anti-S.aureus 1/10 and 50 μl 1/100 and protein 1/100 1/1000

For flow cytometric analysis, the human PMNs are first identifiedaccording to their size and granularity and then verified by surfaceexpression of specific markers (CD35, CD16, GR1, etc.), then oxidativeburst marker. The data are given in terms of percent of activated hPMNsable to induce oxidative burst in comparison with the negative controlgroup. (Didier, 2003; Ploppa, 2008).

Example 39 Opsonophagocytic Assay

An opsonophagocytic assay (OPA) has been developed to estimate thefunctional phagocytic activity of serum for Staphylococcus aureus bymeasuring the complement dependent opsonic activity of the serum. TheOPA is summarized on Table 17. Two strains of S. aureus are used in theassay. Strain DU5875, which does not produce capsule or protein A, isused to best control the assay. Strain LST4 Lowenstein, which doesexpress capsule and protein A, is used as a wild-type strain in theassay. The number of bacteria used in the assay is dependent on thesource of the effector white blood cells and ranges in concentrationfrom 1×10⁵ cfu/ml to 5×10⁷ cfu/ml. White blood cells from healthy humanvolunteers or from a human promyelocytic leukemia cell line, HL □60, areused as phagocytic effector cells. As Table 17 indicates, the number ofeffector cells used in the assay is dependent on the source of thecells. Baby rabbit serum is used as a source of complement and isdiluted from 1% to 10% depending on the lot of serum in order tomaximize the functional complement activity of the rabbit serum whileminimizing the toxicity. Sera undergoing testing for opsonic activity,pre- or post-immunization, are decomplemented at 56° C. for 30 minutesand are tested in the assay at a dilution of 1:20 to 1:2,000,000.Phagocytosis is determined by viable counts (cfu) of S. aureus. Testserum which demonstrates a significant loss of cfu in combination withactive complement when compared to preimmune serum, is consideredopsonic. The assay data are analyzed by the Student's t-Test usingone-tailed distribution with unequal variance. (Kim 2010; Stranger-Jones2006; Dryla 2005).

TABLE 17 Reagent Final Concentration Live approx. 1 × 10⁵ cfu/ml whenusing HL-60 effector cells Bacteria approx. 5 × 10⁷ cfu/ml when usinghealthy human WBCs Effector HL-60 human cell line chemically induced todifferentiate with cells N,N-dimethylformamide diluted to 10 × 10⁶/ml orhuman white blood cells from healthy volunteers diluted to 1 × 10⁷/mlCom- Baby rabbit serum diluted from 1% to 10% according to Lot ofplement serum Test Serum obtained after the last immunization,decompelemented Serum and serially diluted from 1:20 to 1:2,000,000

Example 40 Immune Mechanism Studies (In Vivo)

In another example, the immune mechanism by which vaccine proteinsconfer protection to mice will be assessed. These experiments caninclude two types: (1) using gene knockout mice in vaccine-challengeexperiments to determine whether specific immune components arenecessary for protection; and (2) adoptive transfer experiments, whereimmune cells from immunized donor mice are transferred into naïverecipients prior to bacterial challenge, in order to test whether thetransferred components are sufficient to confer protection.

For examples involving vaccine challenge experiments in gene knockoutmice, the mice can be purchased from commercial vendors and can includeseveral well characterized strains such as, for example, B cellknockouts (μMT), T cell knockouts (TCRα), and a variety of cytokineknockouts, such as IFN-γ, IL-1α, TNFα, IL-17, etc.). The mice can beimmunized as described in Example 3, and then challenge with S. aureus,as described in Example 5. The use of wild type mice with the samegenetic background (Balb/c) can provide suitable controls to measure theeffect of the knockout on vaccine-mediated protection against S. aureus.For example, if the vaccinated B cell knockout mice die more rapidly, orin larger numbers relative to the vaccinated control mice in response tobacterial challenge, it can be concluded that B cells (or theirproducts) are important for the vaccine-mediated protection against S.aureus. These strategies are standard practice in the field formeasuring contributions of various immune components to vaccineprotection (Spellberg, 2008; Lin, 2009).

For examples involving adoptive transfer of immune components, wild typeBalb/c donor mice can be immunized as described in Example 3 in order togenerate tissue for adoptive transfer. These mice can then be euthanized2-4 weeks after the second immunization and the blood collected viacardiac puncture and secondary lymphoid tissue (lymph nodes and spleen)collected. Lymph nodes collected can include: axillary, brachial,mesenteric, inguinal, superficial cervical, deep cervical, and lumbar.Serum can be isolated from the blood using standard methodology, such ascentrifugation-based serum separators, and then transferred back into aseparate set of recipient mice via intravenous or intraperitonealinjection. The use of 1-3 donor mice per recipient is a reasonable ratiofor serum transfer, and can occur in volumes up to 0.5 ml. In addition,T cells from the donor lymphoid tissue can be purified using antibodiesand magnetic bead enrichment technology (Miltenyi Biotec) that isstandard practice in the field. It is typical to achieve 95-99% cellpurity using these methods, as assessed by staining the cell surfaceproteins with antibodies to specific lineage markers and examining thecells by flow cytometry. The cell populations of interest (e.g., CD4⁺ Tcells, CD8⁺ T cells, etc.) can be transferred back into recipientanimals via intravenous injection (between 2,000,000 and 5,000,000 Tcells per recipient). Recipient mice can receive T cells (or subsets ofT cells), immune serum, or both.

As a negative control, placebo-immunized animals can also be used forserum and T cell isolation followed by transfer back into naïverecipients. As a positive control, a group of recipients that getsimmunized with the standard protective vaccine can be included in orderto provide a baseline assessment of protective efficacy upon bacterialchallenge.

Once the recipient mice have received various transferred cells or sera,they can be challenged with S. aureus as described in Example 5. Basedon the percentage of death and the rate of death, the relativecontribution of various immune components to vaccine protection can beassessed. For instance, if recipients that have been administered Tcells from vaccinated donors are protected against challenge at the samerate as the positive control, it can be concluded that T cells aresufficient for vaccine-mediated protection. This experimental strategyis standard practice in the field for assessing the immunologicalmechanisms of vaccines (Spellberg, 2008; Lin, 2009).

Example 41 Inhibition of Iron Uptake Assay

For assessing whether antibodies directed against SIRP componentsinhibit cell growth by blocking iron uptake, an iron uptake/transportassay can be run using bacterial cells that are pre-incubated withanti-SIRP antisera. S. aureus cells from any strain are grown overnightin chelated or iron-rich media. Cells are subcultured in the same mediaand grown to mid to late log phase, pelleted and incubated withanti-SIRP antisera or control antisera in PBS for up to one hour. Cellswere then harvested by filtration using 0.45 μM filters, thenresuspended in Chelex-100 treated minimal medium to eliminateenvironmental iron. Cells are shaken briefly. Meanwhile, an iron source(for example, ferrichrome) is mixed with radiolabeled ⁵⁵FeCl₂ (oranother radiolabeled iron molecule) with nitrilotriacetic acid andallowed to incubate for several minutes.

To commence iron uptake, a small aliquot (for example, 10 μl) of theradiolabeled iron mixture was added to cells (in a 1 ml volume) in a 10ml Fe-free culture tube. The tube is incubated with periodic vortexingand sampling of aliquots filtered onto membrane filters and washed withLiCl. Following drying, membranes are counted in scintillation fluid toquantify the iron uptake. Cells pre-incubated with anti-SIRP antiserashould be slower to take up iron than cells pre-incubated with controlantisera (Sebulsky, 2000; Goel, 2001).

Example 42 High-Yield Protein Purification Protocol

In some instances, recombinant rSIRPs were purified using a high-yieldmethod. This method is optimized for higher yields and higher purity ofthe polypeptide. The method is performed by resuspending the bacterialpellet containing the recombinant-produced polypeptide in 20 mM Tris pH9; 300 mM NaCl complemented with lysozyme (100 μg/ml final) and MgCl₂ (1mM final). The sample is then incubated with gentle rocking for 15minutes at 4° C. After 15 minutes, 1U/ml of benzonase is added and thesample is incubated for one hour at room temperature. The soluble lysateobtained after centrifugation (20,000×g, 20 minutes at 4° C.) should befiltered with a 0.45 μm filter and added to an equilibrated 5 ml gravitycolumn packed with nickel-His binding resin (Novagen 69670-4, EMDChemicals, Inc., Gibbstown, N.J.). The purification of theN-His₆-protein is performed following manufacturer's instructions. Thepurification of the N-His₆-protein is finalized using a HiLoad 26/60Superdex 75 prep grade size-exclusion column (GE Healthcare Bioscienes,Piscataway, N.J.). The column is equilibrated with 20 mM Tris pH 9; 300mM NaCl using a BioCAD FPLC (Applied Biosystems Inc., Foster City,Calif.) and 15 ml of the protein sample is loaded onto the HiLoad columnat a flow-rate of 1.5 ml/min. The sample is eluted following 3.5 hoursof run time at a flow-rate 1.8 ml/min using 20 mM Tris pH 9; 300 mM NaClas the mobile phase.

The polypeptide is quantified using a modified version of the BCA(Thermo Fisher Scientific, Inc., Rockford, Ill.) procedure where 15%sodium dodecyl sulphate (SDS), 8M urea, and 2.5%3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate(CHAPS) is used to ensure complete solubility of all protein within thesample. The assay also consists of 5% SDS added to the BCA workingreagent to maintain solubility of the protein during the BCA reactivephase. The reading and analysis of the BCA is performed according to theproduct literature.

For densitometric analysis, 3.0 μg of final product antigen wasquantified for purity using 10% SDS PAGE, stained with coomassie andimaged using an ODYSSEY scanner (LiCor Biosciences, Lincoln, Nebr.). Thestained gel was scanned and the areas of the major components weredetermined relative to the total area. Residual Endotoxin was removedusing an Endotrap blue one/endosafe kit (Hyglos GmbH, Regendburg,Germany). The final batch is stored with a concentration range of 1.0mg/ml to 4.0 mg/ml in PBS storage buffer at less than −70° C. inappropriate aliquots.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1. A composition comprising: an isolated polypeptide having at least 92%sequence similarity to the amino acid sequence of SEQ ID NO:397, withthe proviso that if the isolated polypeptide includes one or moreadditional amino acids at the amino terminal, the one or more additionalamino acids include at least one amino acid deletion or at least oneamino acid substitution compared to amino acids 1-26 of SEQ ID NO:399.2-57. (canceled)